Enterosalivary nitrate metabolism and the microbiome: intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health

Abstract

Recent insights into the bioactivation and signaling actions of inorganic, dietary nitrate and nitrite now suggest a critical role for the microbiome in the development of cardiac and pulmonary vascular diseases. Once thought to be the inert, end-products of endothelial-derived nitric oxide (NO) heme-oxidation, nitrate and nitrite are now considered major sources of exogenous NO that exhibit enhanced vasoactive signaling activity under conditions of hypoxia and stress. The bioavailability of nitrate and nitrite depend on the enzymatic reduction of nitrate to nitrite by a unique set of bacterial nitrate reductase enzymes possessed by specific bacterial populations in the mammalian mouth and gut. The pathogenesis of pulmonary hypertension (PH), obesity, hypertension and CVD are linked to defects in NO signaling, suggesting a role for commensal oral bacteria to shape the development of PH through the formation of nitrite, NO and other bioactive nitrogen oxides. Oral supplementation with inorganic nitrate or nitrate-containing foods exert pleiotropic, beneficial vascular effects in the setting of inflammation, endothelial dysfunction, ischemia-reperfusion injury and in pre-clinical models of PH, while traditional high-nitrate dietary patterns are associated with beneficial outcomes in hypertension, obesity and CVD. These observations highlight the potential of the microbiome in the development of novel nitrate- and nitrite-based therapeutics for PH, CVD and their risk factors.

Graphical Abstract

Introduction

The study of the human microbiome has had a profound impact on the perception of human health. Characterization of body site-specific microbial ecosystems has revealed a highly diverse and regional set of microbial community networks that can exert systemic effects on the host through the production of metabolites, shaping the immune response, and influencing host gene expression[1]. Changes in these microbial populations have been linked to many ‘non-infectious’ diseases, such as obesity, diabetes, and cardiovascular disease (CVD). As research progresses and the microbiome is increasingly implicated in a wide range of diseases, the focus of work has shifted to the elucidation of the mechanisms behind such associations and their translation into tangible therapeutic solutions.

Despite an explosion of associative studies involving the microbiome and vascular and cardiovascular diseases, few connections have been identified with mechanistic certainty[2,3]. For example, the contribution of the microbiome in the development of pulmonary hypertension (PH) is emerging as a critical mitigating factor. Deficits in mammalian nitric oxide (NO) signaling have been linked to PH pathogenesis, as well as a host of associated risk factors such as hypertension, obesity, reduced insulin sensitivity and CVD. Further, recent insight into the biochemical reactions and signaling actions of nitrate, nitrite and nitric oxide reveal that the bioavailability of inorganic nitrate and nitrite, the major sources of exogenous NO, is dependent on the metabolic activity of specific oral microbiota. Thus, there is encouraging evidence for the study of microbe-driven, nitrogen oxide pharmacology in the treatment of several forms of PH.

Herein, we discuss a novel interrelationship between mammalian NO biology and microbial control of nitrate and nitrite bioavailability in the development of PH. Our discussion centers on the hypothesis that maintenance of pulmonary and systemic vascular health in mammals in part depends on the enzymatic reduction of dietary nitrates by commensal oral bacteria to vasoactive and anti-inflammatory mediators that provide systemic NO-effects critical under conditions of hypoxemia and stress. We review the evidence supporting a beneficial role of dietary and salivary inorganic nitrogen salts in the oxygen-independent generation of NO and secondary reaction products important for the maintenance of pulmonary vascular health, with a particular focus on the critical role of oral and gut microbial communities in the enzymatic reduction of nitrate to nitrite and the impact of diet and probiotics.

Dysfunctional Nitric Oxide Signaling underlies Pulmonary Hypertension Pathogenesis

Pulmonary Hypertension

Pulmonary hypertension (PH) is a progressive and fatal clinical disorder of the pulmonary circulation characterized by increased pulmonary vascular resistance, elevated pulmonary pressures (mean pulmonary arterial pressures ≥25mmHg), right ventricular overload and ultimately collapse[4,5]. There are a multitude of causes of PH. Many cases are idiopathic (Group I-pulmonary arterial hypertension, PAH), and secondary PH occurs in certain subsets of patients in the setting of left heart dysfunction (Group II) or secondary to advanced lung diseases (Group III)[5,6]. Other conditions such as HIV infection are classified as Group I, but may also have unique attributes[7–11]. Despite the diversity of patients who develop PH, many share a common link to the metabolic syndrome[4,12–14]. For example, obesity, hyperlipidemia, and systemic hypertension are common risk factors for Group I PAH as well as Group II (secondary to left-sided heart failure or PH-heart failure with preserved ejection fraction, HFpEF)[12,15,16], and are also a feature of HIV infection[17,18]. It is therefore likely that on some level, PH can be characterized by similar pathogenic mechanisms.

Nitric Oxide Signaling Dysfunction in Pulmonary Hypertension

NO is an endogenously-produced, lipophilic, and diffusible molecule that exerts a diverse array of critical autocrine and paracrine signaling activities. Chiefly, NO acts directly on smooth muscle cells via activation of soluble guanylyl cyclase leading to formation of the second-messenger, cyclic guanosine monophosphate (cGMP), that in turn promotes smooth muscle relaxation and inhibits both platelet function and vascular smooth muscle cell proliferation and migration[19–21]. Classically, the majority of NO is derived from the oxidation of the essential amino acid, L-arginine by a set of 3 isoforms of the enzyme nitric oxide synthase (NOS) in a reaction requiring oxygen, reduced nicotinamide-adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin (BH4) and other cofactors[20,22]. Importantly, endothelial-derived NO production and function is largely determined by, and integral to the maintenance of a healthy endothelium[23–25].

Pathologically, all types of PH develop via an early, common vasculopathy driven by disproportionate pulmonary vasoconstriction and dramatic remodeling of the endothelium and vessel wall[5,26,27]. The hallmark feature of PAH and other forms of PH is reduced bioavailability of NO signaling and poor endothelial responsiveness to triggers of vasodilatation[28–34]. Similarly, vascular disease, cardiovascular disease (CVD), and metabolic syndrome all share effective loss of NO function[23,35–39]. In PH, the cause of NO signaling loss remains unclear, possibly being related to abnormal endothelial NOS expression[40], impaired NO formation[41–43] and increased NO scavenging by elevated rates of reactive oxygen species (ROS) generation by mitochondria, oxidases, oxygenases and uncoupling of NOS electron transfer reactions[23,44–47]. Given that most efficacious therapies for the treatment of PH involve targeting of NO signaling[48,49], it is clear that pulmonary vascular health is intimately tied to effective NO bioactivity.

The nitrate-nitrite-NO Pathway & Enterosalivary Circulation of Nitrate as ‘Alternate’ Source of NO Signaling

Nitrite, derived from the oxygen- and NOS-independent, microbial enzymatic reduction of dietary nitrate, has been identified as an important secondary source of NO and other bioactive nitrogen oxides[25]. Nitrite metabolism can be activated by hypoxia, low pH and reactions with metalloproteins[50–58]. As with NOS-derived NO, inorganic nitrite demonstrates robust NO-signaling effects[52,53,57,59] and contributes to basal vascular tone[51,55,60,61], blood pressure[62–66], protection from endothelial dysfunction[67–71] and vascular inflammation[72] under conditions of metabolic and inflammatory stress.

Formation of nitrite and propagation of its downstream NO-signaling effects depends on the oral bacterial reduction of inorganic nitrate by a set of bacterial nitrate reductase enzymes (NaRs) that are largely absent from the human genome[73–78]. In a process referred to as the enterosalivary nitrate circulation, or the nitrate-nitrite-NO pathway, dietary nitrate is swallowed and rapidly absorbed in the proximal gastrointestinal tract (Figure 1)[79–81]. In proportion to the dietary load of nitrate, approximately 25% of total circulating nitrate is actively sequestered into salivary glands and concentrated in saliva up to 20 times that in plasma[73,75,82–88]. Once in the mouth, salivary nitrate undergoes NOS- and oxygen-independent, enzymatic reduction to nitrite. Nitrite is then swallowed where it can undergo a diversity of downstream reactions, including formation of NO and NO-donating species following a) protonation to HNO₂ and an array of secondary nitrosating and nitrating species in reactions facilitated by the low pH of the stomach[80,83,89–91], or b) further reduction by enteric bacterial nitrite reductases (NiRs)[74]. These reactions also lead to the generation of electrophilic nitro-fatty acid (NO₂-FA) signaling mediators, including NO₂-conjugated linoleic acid (NO₂-CLA) and S-nitrosothiol derivatives, all of which can be detected at increased concentrations in the systemic circulation following oral consumption of nitrate[72,92–96]. In turn, these reactions and their products can not only instigate salutary NO signaling responses, but also post-translational protein modifications and altered patterns of gene expression. These reactions can beneficially regulate cardio-metabolic function and will be attenuated or abolished by chemical or physical destruction of the oral microbiota[60,75,76,97–99].

Figure 1.

Schematic of the enterosalivary nitrate (NO₃⁻), nitrite (NO₂⁻), nitric oxide (NO), and nitro-fatty acid (NO₂-FA) pathways and interactions with the oral and gut microbiome.

Formation of and signal transduction by nitrate-derived nitrite, NO and bioactive nitrogen oxides is not a linear progression to NO and cGMP-dependent signaling, as the reference to the enterosalivary nitrate-nitrite-NO pathway might suggest. There exists a broad range of redox-derived nitrogen oxides that exert both cGMP-dependent and -independent signaling actions as a result of nitrosative-, nitrative- and oxidative-induced post-translational protein modifications and subsequent changes in protein function and gene expression profiles[100–102]. Several aspects of these reactions are addressed below and have been extensively reviewed previously[91,93,96,103–105].

Microbial Nitrate Reduction to Nitrite and NO is Physiologically Important

Given the importance of NO’s effects on the vasculature and the apparent contribution of the enterosalivary nitrate pathway to systemic NO bioavailability, the loss of the ability to reduce nitrate to nitrite and NO is expected to have systemic implications, particularly in situations of systemic or vascular metabolic and inflammatory stress. Extensive evidence supports that microbial bioactivation of nitrate may play a role in the prevention and treatment of systemic vascular pathology and PH.

Blood Pressure & Endothelial Health

An impressive body of preclinical and clinical evidence has demonstrated beneficial blood pressure and vascular effects of nitrate and nitrite supplementation in the form of sodium/potassium salt or concentrated dietary supplement such as beetroot. Studies in Sprague-Dawley rats[78] and normal human volunteers[106] showed that depletion of oral bacterial nitrate reductases by chlorhexidine mouthwash correlated with a 90% decrease in oral nitrite levels (p<0.001) in humans, along with a 25% decrease in plasma levels (p=0.001), and 2–3.5mmHg increase in blood pressure that occurred in proportion to decreased plasma nitrite (R²=0.56, p=0.002)[106]. At least 19 studies in hypertensive, overweight, and other patient populations have been performed, all revealing predictable acute or chronic blood pressure reduction with varying types of nitrate supplementation[107], and that antimicrobial mouthwash abolishes this beneficial effect both acutely and chronically (reviewed in:[108,109]).

From the standpoint of vascular health, endothelial dysfunction has been associated with decreased circulating levels of nitrite[67,110]. Oral supplementation with either nitrite or nitrate increases circulating nitrite levels and prevents and reverses endothelial dysfunction in a dose-dependent manner both in mice[68,69] and in healthy[70,71,97,111,112], hypertensive[113], and obese individuals[114], or in those with hypercholesterolemia[115] and peripheral arterial disease[116]. Further, dietary nitrate and nitrite confer protection from intimal hyperplasia in rodents[117–119], while nitrate supplementation prevents or limits ischemia-reperfusion injury and endothelial dysfunction in both animal[98,120–124] and human[97,125] models of ischemia-reperfusion.

Exercise Capacity & Oxygen Utilization

Microbial reduction of nitrate to nitrite, NO and other bioactive nitrogen oxides may influence more than vascular health alone. Functional resilience to debilitating diseases such as PH may also be conferred by microbes via improved exercise capacity, oxygen utilization, and myocardial function[126]. For example, one study demonstrated that nitrate supplementation offers net decrease in oxygen cost (V_O2, mL/kg/min) during moderate and heavy submaximal exercise in healthy adults[127], an effect that over 14 studies have confirmed with reduced V_O2 and increased work capacity in response to nitrate during submaximal exercise in healthy individuals[128,129] (reviewed in:[130,131]) and in certain chronic diseases[127,132]. Additionally, in the setting of heart failure and cardiomyopathy, dietary nitrate improves left ventricular function in mice[133] and overall cardiac contractility and exercise performance in humans[134–137], suggesting the critical potential of exploiting the oral microbiome to enhance functional outcomes in patients via mechanisms spanning from improved mitochondrial function to increased vascular and cardiac performance[138].

Pulmonary Hypertension

In terms of PH, there have been no human clinical studies testing the effects of oral nitrate or nitrite, though animal model data support a therapeutic role. Inhaled NO induces vasodilation in PAH and is capable of reversing hypoxic pulmonary vasoconstriction[139–141]. Similarly, nitrite administered via inhalation or infusion is a selective pulmonary vasodilator with increased activity in hypoxia[142–144], and is capable of inhibiting both hypoxia-induced and monocrotaline-induced PH in rats[145]. Delivered via intravenous, inhaled, or oral routes, nitrite reduces pulmonary and right heart pressures and prevents or reverses characteristic vascular remodeling and right ventricular hypertrophy (RVH) in several animal models of PAH[15,117,143,146–149]. In a single experiment using nitrate, dietary supplementation prevented pulmonary vascular remodeling and RVH in both wild-type and endothelial NOS-deficient mice[150], indicating an active role for microbial, nitrate-derived nitrite and NO in the development and progression of PH. Further, in a population of individuals with HFpEF, there was a 14% increase in cardiopulmonary exercise testing performance in 17 patients after a single dose of beetroot juice (12.9 mmol nitrate)[137], and in another study, a 24% improvement in submaximal aerobic endurance with 1 week of daily beetroot juice consumption (6.1 mmol nitrate)[151]. These studies support the clinical utility of nitrate in at least some types of PH.

Current evidence suggests the biological importance of nitrate and its enzymatic and non-enzymatic reaction products in maintenance of vascular health and PH. These data also support the concept that an intricate and dependent relationship exists between mammals and commensal, oral, nitrate-reducing microbiota in maintaining adequate NO signaling. Yet, despite the importance of these microbial populations, relatively little is known about the identity and function of specific bacteria and their effects on nitrate metabolism.

Safety of Inorganic Nitrate and Nitrite

For many years, dietary nitrate and nitrite were considered harmful for regular human consumption due to two prominent health concerns, the development of methemoglobinemia and the potential for carcinogenic effects. Ostensibly, the toxic effects of nitrate and nitrite are driven by the more reactive nitrite after either oral reduction by the enterosalivary pathway or direct consumption. In the acute setting, nitrite can bind to hemoglobin and oxidize the ferrous iron in heme to the ferric state, thereby forming methemoglobin (metHgb) and preventing the binding of oxygen[152]. Methemogobinemia becomes clinically significant when baseline levels of metHgb (1–3%) rise to 5–12%, with cyanosis and fatal toxicity occurring at levels between 30–50%[153]. Early concerns of infant exposures led to current regulations limiting nitrate content in water to 44 mg/L[154,155], although more recent evidence in both infants and adults suggests that exceptionally high concentrations of nitrite (and well beyond the <75mg nitrite necessary to achieve blood pressure effects) would be necessary to pose a significant threat to safety[25,51,72,113,155–158]. Similarly, little evidence exists to suggest that chronic nitrate or nitrite consumption are directly carcinogenic in humans[159,160]. However, in certain settings nitrite can form nitrous acid via the N-nitrosation pathway in the stomach, which then reacts with dietary amines and amides to form powerful alkylating, and carcinogenic, N-nitroso compounds[155,161,162]. Indeed, nitrites in combination with dietary amines or amides, such as in red and cured meats, have been linked to stomach and several other cancers[155,157,160,163–166]. Excitingly, when ingested nitrate and nitrite are rather paired with plant- and fruit-based polyphenols and essential nutrients such as ascorbate, gastric nitrosation is inhibited[162,167,168], suggesting that the mutagenic potential of dietary nitrate and nitrite is likely contextual, depending on the complex interactions between nitrate and nitrite with factors such as the relative content of amines, amides and plant-based, antioxidant substrate[155,169].

The Oral Microbiome and Bacterial Nitrate Reduction

Complexity of the Oral Microbiome

As the body’s threshold to the outside world, the oral cavity is one of the most ecologically dynamic, and most studied, environments in the body. Culture-independent sequencing of the bacterial 16S ribosome has suggested an estimated 50–100 billion bacteria in the mouth, comprised of nearly 700 identified bacterial species[170,171]. Up to 80% of oral bacteria are dominated by about 200 species in the Phyla Firmicutes (esp. Streptococcus, Veillonella, Granulicatella, Gemella spp.) and Proteobacteria (esp. Neisseria, Haemophilus spp.), with contributions from Bacteroidetes, Actinobacteria and Fusobacteria totaling upwards of 95% of all identified oral microbiota (Figure 2)[171–177]. Further diversity exists between the varied micro-ecologies of the mouth[174,178], such as the tooth surface, gingiva, hard and soft palate, and even regionally on the tongue (Figure 2)[173,176,179]. Local variation in site-specific “core” microbial members speaks to the heterogeneity of influences bacterial communities experience, including host factors such as age, diet, geography and oral health[180–182].

Figure 2.

The ‘core’ oral microbiome. Charts represent a general summary of the relative abundances of major bacterial phyla (>1% total microbial abundance) constituting the normal microbiome of the human oral cavity. Individual charts represent regional microbial diversity within the entire oral cavity, saliva and oral wash, dorsum of the tongue, buccal mucosa, subgingiva, and the hard palate. Values presented are summary estimates only, extrapolated and pooled from multiple published analyses[173–175,177,194].

Amid this complexity, an ever-growing number of specific microbial taxa have been associated with both oral and systemic diseases[181,182]. For instance, saccharolytic (sugar metabolizing) bacteria such as Streptococcus and Lactobacillus have been associated with dental caries, while proteolytic (protein metabolizing) bacteria such as Prevotella and Porphyromonas have been associated with periodontitis and halitosis[183]. Associations between atherosclerosis and Bacilllus typhosus were first described over 125 years ago[184], while more recent work has linked Porphyromonas gingivalis to atherosclerosis[185], smoking[186], and several cancers[187]. Yet, individual bacteria do not act in a vacuum, but rather function in interdependent microbial communities whose conglomerate activity can impact oral and systemic health[183,188,189]. Indeed, even the nitrate-nitrite-NO pathway is affected by microbial diversity[190] and oral pathology[191–193]. It is within this context of regional, community function that the importance of oral bacterial nitrate reduction to nitrite, NO and other bioactive nitrogen oxides must be viewed.

Bacterial Nitrate Reduction

Bacterial metabolism of inorganic nitrogen compounds, ranging from nitrate in its most oxidized state to ammonia in its most reduced, is fundamental to the global/ecological nitrogen cycle. Reduction of nitrate to nitrite by molybdenum-dependent nitrate reductases is the initial and rate-limiting step in anaerobic nitrogen metabolism[77]. Broadly, nitrate reduction is classified into three major pathways (Figure 3)[77,195–197]: 1) Respiratory denitrification, catalyzed by membrane-bound nitrate reductase, nar, or periplasmic nap, is the major electrochemical respiratory pathway for bacterial energy generation under hypoxia via generation of a proton motive force, serving as a two-electron sink in the final step of ATP generation via the respiratory electron transport chain[196]. In this process, nitrite is further reduced to gaseous NO, nitrous oxide, and dinitrogen by a series of reducing enzymes, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively. Gaseous nitrogen can then either be excreted or, in a subset of bacteria, undergo further reduction via the enzyme nitrogenase to ammonia, in a process called nitrogen fixation. 2) Dissimilatory nitrate reduction to ammonia (DNRA), catalyzed by periplasmic nitrate reductase, nap, is thought to be the major detoxification/excretion pathway for nitrogen compounds in which nitrite is directly converted to ammonia and excreted[198]. Alternatively, dissimilatory nitrate reduction can indirectly participate in ATP generation when coupled with formate oxidation[77,199]. 3) Assimilatory nitrate reduction, catalyzed by cytoplasmic nitrate reductase, nas, is the biosynthetic, anabolic pathway by which nitrite is further reduced to ammonia, which can then undergo ammonium assimilation via production of the amino acid glutamine and incorporated into the bacterial biomass.

Figure 3.

Schematic representation of major bacterial nitrate reduction pathways. Abbreviations: DNRA, dissimilatory nitrate reduction to ammonia; ATP, adenosine triphosphate; NO₃⁻, nitrate; NO₂⁻,nitrite; NO, nitric oxide; N₂O, nitrous oxide; N₂, dinitrogen; NH₃, ammonia; NH₄^•, ammonium; glu, glutamine; nar, nap, and nas, nitrate reductases; nir, nrf, nitrite reductases; nor, nitric oxide reductase; nos, nitrous oxide reductase; nif, nitrogenase; e⁻, electrons. Adapted from Sparacino-Watkins et al.[77] with permission of The Royal Society of Chemistry. http://dx.doi.org/10.1039/c3cs60249d.

Numerous bacteria contain genomic nitrate reductase genes and are capable of their expression and of reducing nitrate to nitrite in culture; however, in vivo identification of the major nitrate-reducing bacteria that contribute to the mammalian enterosalivary nitrate pathway is less certain. The bulk of physiologically relevant nitrate reduction occurs via the activity of facultative anaerobic bacteria dwelling in crypts of the posterior dorsum of the tongue[81,200,201]. In both rat and human studies species of Firmicutes (Staphylococcus, Streptococcus and Veillonella) and Actinobacteria (Actinomyces) have been most frequently identified as among the highest nitrate-reducers, while numerous other taxa have also been identified, including Pasteurella, Rothia, Neisseria, Haemophilus, Granulicatella, etc.[190,200–204]. Using nitrate supplementation as selective pressure for bacteria capable of nitrate reduction, several studies found proliferation of Veillonella[205], Streptococcus[204], Neisseria[115], Haemophilus[204], and Rothia species[115], all previously identified as high nitrate reducers. Yet, despite these varied attempts, there remains a high degree of variability in results depending on numerous methodological factors[206], including source and sampling of sample (saliva versus oral wash versus tongue scraping), direct sampling versus culture, and choice of 16S variable region for sequencing.

Also critical to bacterial nitrate reduction is the trans-membrane transport of nitrate and subsequent management of potentially cytotoxic nitrite. Nitrate reductase subtypes are found in locations commensurate with their function: membrane-bound respiratory nar faces the cytoplasm, facilitating movement of protons across the cell wall and thereby creating the proton motive force necessary for ATP generation; membrane-bound dissimilatory/detoxifying nap faces the periplasm, allowing for protective neutralization of excess nitrate before it is transported into the cell; and soluble assimilatory nas moves freely within cytoplasm, close to active sites of biosynthesis. Depending on enzyme location, nitrate transport is thought to occur either by direct, ATP-hydrolyzing, ABC-type transporters in assimilatory nitrate reduction, or via a set of secondary nitrate/nitrite porters (NNPs) of the major facilitator superfamily (MFS), NarK and NarU, in respiratory nitrate reduction [207,208]. Of biological importance, however, two functionally distinct NarK subfamilies are thought to represent the bulk of nitrate-specific transport[208,209]. While the precise mechanism of transport is not yet known, several structural analyses have suggested that transporter NarK functions as an electroneutral nitrate/nitrite antiporter, exchanging periplasmic nitrate one-to-one for cytoplasmic nitrite[208,210], whereas NarU acts as a nitrate/proton symporter, linking nitrate uptake to an existing proton gradient[211,212]. Notably, control of transporter gene expression is linked to the same oxygen- and nitrate-responsive regulatory systems used by the nitrate reductases[207,213], reinforcing the concept that bacterial nitrate reduction is a dynamic process intimately related to both bacterial genetics and local environmental pressures.

Local Factors Influence Bacterial Nitrate Reduction

From the perspective of bacterial survival and the development of targeted therapies, several additional features can influence bacterial nitrate reduction. Microbiota are fundamentally communal organisms and exist in mutualistic, interdependent networks that are crucial to their survival. These networks allow adaptation to host and environmental pressures through the sharing of metabolic products, adhesion and biofilm formation, chemical signaling, quorum sensing, and horizontal gene transfer (reviewed in:[214,215]). Based on genomic content, it is possible for a single bacterial strain to fully reduce nitrate to NO, nitrogen, or ammonia via multiple reductive pathways. However, the presence and expression of nitrate reductase genes is highly variable within and between bacterial species[216]. Demonstrating the complexity of community metabolism, an ex vivo experiment showed increased nitrate consumption in a polymicrobial culture of mixed high- and low-nitrate reducing bacteria challenged with nitrate compared to one of high-nitrate reducers alone[190]. Similarly, there was decreased NO generation in a polymicrobial culture containing NO-consuming bacteria in rats[217]. Thus, alterations in bacterial communities may be more important than changes in single bacterial species, and due consideration must be paid to functional community networks in the design and implementation of targeted therapies[218,219].

Further, bacterial networks and host-associations must include the robust viral and fungal flora that also cohabit the mouth. Bacteriophages share with host bacteria a similar site-specific diversity in the mouth and disease associations such as periodontitis[220,221]. Other viruses, such as HIV, also alter the oral microbiome, mycobiome, and NO-driven disease susceptibility (such as CVD and PH)[222–224]. They also exert synergistic effects with bacteria, as with herpesviruses in the pathogenesis of periodontitis[225]. Fungal populations also share regional diversity and complex metabolic interactions with resident bacteria[226,227] and display multiple disease associations[228]. Importantly, multiple fungal species, including the ubiquitous Candida, contain a fungal nitrate reductase enzyme and are known nitrate-reducers in the environment[229,230]. Both the oral virome and mycobiome thus have the potential to alter bacterial nitrate metabolism through alterations in bacterial populations and their metabolism, direct reduction of nitrate and alterations in metabolic substrate, changing local host and environmental conditions such as stimulating inflammation, and propagation of oral and systemic disease.

Second, protective host factors may exert selective environmental pressures that influence mechanisms of oral microbial metabolism of nitrogen. For instance, nitrite and nitrate are important protective elements of the oral and mucosal immune response, especially in settings of stress and periodontal injury[86,193,231]. Nitrite can exert pH-dependent antimicrobial effects[204,232,233] via formation of reactive secondary nitrogen oxides that can disrupt aerobic respiration[234], inhibit bacterial acid production, and disrupt biofilm formation[235–237]. In settings of low pH and high nitrate and nitrite concentrations, selected bacteria and bacterial communities capable of nitrate reduction may preferentially survive or expand their population[115,205]. Indeed, increased production of the weak base, ammonia via nitrate reduction is a well-known strategy for bacteria to alkalinize their cytoplasm and local environment and protect against the potential toxic effects of high concentrations of nitrate, nitrite and NO[238]. This may relate to the paradoxical association between nitrate-reducing oral bacteria and protection from dental caries[191,205,232,238,239].

Third, it is clear that pH, particularly with regard to stomach acidity, plays an important role in nitrate, nitrite, and NO metabolism. One of the main functions of low gastric pH is to clear pathogenic bacteria before they enter the distal GI tract. Mechanistically, much of the toxicity is due to the non-enzymatic protonation of nitrite to HNO₂ and NO and subsequent oxidative and nitrosative stress. A prominent example of such pH-NO and host-microbe dynamics is the well-studied human pathogen Helicobacter pylori. Epidemiologically, H. pylori is linked to peptic ulcer disease, gastric cancer, as well as diabetes, autoimmune disease, chronic inflammation, CVD and endothelial dysfunction[240–245]. To survive the hostile gastric milieu, H. pylori has developed robust mechanisms for altering its local environment on two fronts. First, H. pylori has been shown to inhibit iNOS expression and NO production by the host macrophage[242]. Second, H. pylori can alter stomach pH via expression of urease that allows for the formation of both ammonia and bicarbonate from urea, thus reducing the acidity of the stomach and potentially reducing the nascent pH-dependent formation of NO from salivary nitrite[242,246,247]. Supporting the importance of such mechanisms, a growing body of evidence has linked acid-reducing medications such as proton pump inhibitors and histamine H₂ antagonists, with increased risk of enteric infections such as clostridium difficile[248–250] and profound alterations of the gut microbiome[251–253]. Additionally, acid-reduction is known to reduce gastric production of NO[76,90,254] and has recently been shown to attenuate the blood pressure lowering effects of nitrite in healthy adults[255]. These data then suggest that gastric pH may be equally as important in nitrate bioactivation as a viable oral, nitrate reducing microbiome.

Finally, the oxygen content itself in any particular oral or gut microenvironment can profoundly influence microbial mechanisms of respiration and the role of particular nitrate reduction pathways. The vast majority of oral and GI nitrate reducers are facultative anaerobes. From the standpoint of energetics, facultative anaerobes prefer aerobic respiration, although they maintain the capacity to use anaerobic fermentation for growth in anoxic or low oxygen environments, a mechanism dominated by the respiratory nitrate reductive pathway[77]. Indeed, the expression and activation of bacterial nitrate reductases are tightly regulated via oxygen-dependent and nitrate-responsive mechanisms[77,256]. For instance, the respiratory nitrate reductase, nar, expresses two major operons, NarGHI and NarZYW[77,256]. The first, NarGHI, is highly oxygen-responsive, under primary transcriptional control via the oxygen sensor fumarate nitrate reductase regulator (FNR), and appears to be the major contributor to respiratory anaerobic activity[256–258]. Conversely, NarZYW is constitutively expressed at low levels in the aerobic setting and only up-regulated in response to high nitrate concentrations, likely aiding in the transition from aerobic to anaerobic respiration [256,259,260]. Comparatively, while the DNRA nitrate reductase, nap, shares some regulatory elements with nar such as the oxygen-dependent FNR[77,256,261–264], it is notable that nap is maximally expressed under low oxygen and low nitrate conditions, a difference suggested to possibly relate to a selective advantage over nar in the distal GI tract[77,265]. What is clear is that the primary determinants of bacterial respiration are the ambient oxygen tension and nitrate concentration at any given point.

Understanding of the particular conditions that trigger such dramatic shifts in microbial energy production is lso necessary to understand in vivo function. In a recent set of experiments using gut facultative anaerobes, Escherichia coli and several Lactobacillus sp., there was an increase in the expression of nitrate reductase enzymes, an increase in nitrite formation, and a clear growth benefit in the setting of adequate nitrate as oxygen concentration decreases below 4% [233]. Conversely, microbiota isolated from dental plaque were capable of denitrification under aerobic conditions, supporting the idea that biological activity is regulated by multiple forms of nitrate reductase enzymes[192]. Biologically important nitrate reduction, therefore, arises from regional interactions between oxygen tension, nitrate concentration, and microbial genetics.

In the mammalian GI tract, several remarkably steep oxygen gradients must be considered in order to understand the functional context of nitrate reductase activity (Figure 4). In the mouth, regions of high oxygen concentration (20–114mmHg, ~3–15%) are generally expected to be in whole saliva, on the tooth surface, and in dental plaque[266]. Conversely, areas that have high nitrate reductase activity such as the dorsum of the tongue, gingival plaque and periodontal pockets demonstrate low relative oxygen concentrations (8–13 mmHg, ~1–2%)[81,193,201,266]. In the gut, two broad oxygen gradients must be considered as well. Longitudinally, oxygen concentrations generally fall, with the highest pO2 in the proximal gut including the gastric fundus (77mmHg; ~10%) and small bowel (30–45mmHg, ~4–6%), and declining through the colon to concentrations of less than 3 mmHg (ρ%) in the sigmoid colon and rectum[267]. Second, diffusion of oxygen from the enteral vasculature through the submucosal tissue and into the intestinal lumen creates an incredibly steep oxygen gradient, ranging from 80–100mmHg (~10–13%) in the submucosa and base of the villi, down to only 8–10mmHg (~1–2%) in the overlying mucus layer and an essentially anaerobic luminal center (Figure 4)[233,267]. Add to this the wide temporal fluctuations in oxygen tension at the mucosal border during the massive hyperemia experienced after meals, and it rapidly becomes clear that the ability to switch metabolism from aerobic to largely nitrate-driven anaerobic is a requirement for microbial survival[267–269]. Considering, however, that despite the capacity of bacteria in the distal GI tract to reduce nitrate[74], it is unlikely they significantly contribute to the enterosalivary circulation given that the majority of nitrate and nitrite are absorbed in the proximal small intestine[233,270].

Figure 4.

Diagram of estimated oxygen concentration at various locations in the oral cavity and gastrointestinal tract. Left figure identifies locations in the oral cavity, stomach, and large and small bowel. Right figure is representative of a segment of bowel in cross-section. pO₂, partial pressure of oxygen (mmHg). pO₂ estimates from Espey 2013[267] and Hill & Marsh 1989[266].

Many questions remain regarding the mechanisms of microbial nitrate reduction and contributions to enterosalivary-derived nitrite, NO and other bioactive nitrogen oxides. Nitrite is the common early intermediate in all bacterial nitrate-reductive pathways, but it is unlikely that all three mechanisms contribute in equal amounts. In hypoxic conditions, respiratory denitrification is the most energetically favorable process that promotes respiration-dependent growth[77]. The logical assumption would be that in a healthy mouth, ATP-generating respiration would predominate. Conversely, under conditions of host stress or inflammation, selective pressures on the microbiota change, leading to increased nitrate, nitrite and reactive nitrogen oxides, reduced pH, and a hostile environment that may favor the dissimilatory pathway, leading to ammonia production, alkalization, and nitrogen detoxification[271]. Hence, it is likely that different environmental conditions favor different mechanisms of nitrate reduction and their associated microbial communities. Further understanding of this point is crucial to the development of safe and effective therapies that target this pathway.

Dysbiosis of the Microbiome in Pulmonary hypertension & Vascular Disease

The Oral Microbiome and ph

To date, there are no studies that directly link microbial community features of the oral and gut microbiome, nitrate bioavailability, and the development of PH. The critical evidence of such an association, however, rests in the global dependency of enterosalivary nitrate reduction on living oral microbiota to enact its biologic activity[73,99,109]. As reviewed, oral administration of nitrate and nitrite provides dose-dependent improvement in endothelial dysfunction, inflammation and vascular function[71], protection from ischemia-reperfusion injury[97,98,272], and reversal of vascular and ventricular remodeling in several models of PAH[147,150]. Clinically, nitrate supplementation will reliably produce anti-hypertensive effects[107], improved exercise efficiency[127], and improved exercise performance in patients with heart failure and HFpEF[135,137,151]. Based on these findings, there can be little doubt that oral nitrate supplementation exerts profound beneficial vascular effects via microbial-dependent bioactivation in numerous settings closely related to PH and its pathophysiological mechanisms.

From the standpoint of the oral microbiota, there is evidence that particular population changes are associated with pathologies that confer an increased risk of developing PH and other vascular disease, including obesity[273–275], diabetes[276], and insulin resistance[277]. While the mechanistic underpinnings of these effects deserves additional study, it is likely to be transduced in part by NO and the pleiotropic reactions of secondary nitrogen oxides induced by microbial nitrate reduction and downstream reactions. Additional factors could include further gut microbial metabolism of secondary nitrogen oxides, the generation of bioactive fatty acid signaling mediators, and the downstream signaling reactions emanating from a host of other metabolic mediators that could influence PH, especially in metabolic syndrome and obesity[217,233,278,279].

Microbial Communities & Nitrate Bioactivation are Disrupted in Periodontal Disease & Xerostomia

Outside of global shifts in the oral microbiome, two additional aspects of oral health and the microbiome are worth mentioning in relationship to nitrogen oxide metabolism and the development of PH. First, periodontal disease is a condition defined by acute and chronic inflammation of the gingiva that leads to bleeding, reduced clinical attachment, gum recession and eventual tooth loss. Characteristically, disease is accompanied by the transition from symbiotic, facultative microbiota to overgrowth of dysbiotic, anaerobic bacteria together known as the ‘red complex' that reside in the relatively hypoxic subgingival crypts and pockets of the gums[182]. A long-described, yet puzzling and hitherto unproven, association between periodontal disease and CVD[280–282] has been variably attributed to direct vascular entry of these largely nitrate-reducing oral pathogens[283–286], generalized immune activation, systemic inflammation, and endothelial dysfunction, all processes that are improved with periodontal treatment (typically bactericidal chlorhexidine rinses with or without quadrant scaling)[193,287–290]. Interestingly, both salivary nitrate and nitrite concentrations and gingival expression of NO-forming NOS have been found to be increased in periodontal disease, which also tend to increase with disease severity and improve with periodontal treatment[99,193,291,292]. However, it remains uncertain whether increased nitrite derives from oxidation of gingival NOS-derived NO or bacterial reduction of salivary nitrate. What is clear is that inflammation in the localized anaerobic environment of the expanding subgingival crypts, in concert with high salivary nitrate, likely selects for survival and growth of the anaerobic, nitrate-reducing bacteria typical of periodontal disease[193]. Yet, despite these localized, periodontal effects on microbial populations, high salivary nitrite concentrations may also exert antimicrobial effects elsewhere in the oral cavity and gut, possibly disrupting community composition or function of biologically beneficial populations of nitrate-reducing bacteria[204,232–237]. Given these associations and recent links between periodontal disease and PH-associated conditions such as obesity and diabetes[182], it is possible that dysbiosis and systemic inflammation due to periodontal disease and overall oral health may impact or reverse the beneficial effects of physiological enterosalivary nitrate metabolism and promote the development of PH.

An additional and provocative association also exists between dysbiosis of the oral microbiome, PH, and diseases of dysfunctional salivary flow (xerostomia). Nitrate content in saliva is highly regulated[85,86,193,231]. In the setting of salivary gland dysfunction, there is a decrease in salivary nitrate concentration and corollary increase in urinary excretion, thereby reducing total body nitrate content and its enterosalivary circulation[84,193,231,293]. Significantly, several diseases that share both disproportionately high rates of vascular disease and PH, including Sjögren's and systemic sclerosis[294–297], HIV[7,11], and cystic fibrosis[298] also feature dysregulated salivary flow[299–302] and altered oral microbial populations[224,303–306]. While there is no conclusive link between these factors, these associations offer a potentially novel line of investigation.

The Gut Microbiome

Although the major, rate-limiting steps in the nitrate-nitrite-NO pathway occur in mouth, the gut constitutes the nexus of many host-microbe interactions that may impact nitrate recirculation and further reduction to nitrite, NO and other bioactive nitrogen oxides and signaling mediators. Relative to the oral cavity, the gut harbors over 1kg of bacterial biomass and upwards of 1000 different species[307]. Ecological conditions vary immensely along both the length of the GI tract as well as in cross-section from lumen to the mucosal brush-border[173,267,308,309]. Numerous facets of GI microbial community composition and metabolism have been associated with various disease states, including CVD, obesity, diabetes, and features of metabolic syndrome[274,310–313]. There are no studies that directly investigate an association between the gut microbiome, nitrate, and PH. However, several features common to gut dysbiosis, altered NO-signaling, and PH suggest a role in disease including inflammation and NO production.

Inflammation

Inflammation is a prominent feature of PH that may be mediated by the host-microbe interaction[314,315]. Studies suggest that in the gut, greater microbial diversity is associated with protection from inflammation and inflammatory disease[316,317], while conversely, reduced microbial diversity is associated with inflammation, obesity, hypertension, and features of metabolic syndrome[311,313,318,319]. In the injured or diseased gut, inflammation leads to increased production of reactive oxygen species such as superoxide, hydrogen peroxide and hydroxyl radicals and reactive nitrogen oxides such as peroxynitrite, dinitrogen trioxide and nitrogen dioxide[320]. Elevated rates of generation of these oxidative inflammatory mediators can have direct effects on enterocytes and mucosal integrity, impair NO-dependent vascular function, reduce nitrate and nitrite absorption, and exert selective pressure on bacteria by augmenting innate anti-bacterial effects[204,233,321–324]. Consequently, microbial diversity is reduced[325,326] while local and systemic inflammation and decreased NO bioavailability persist, propagating vascular injury and the risk of developing PH.

Recently, the ratio of Firmicutes to Bacteroidetes in the gut has gained increasing acceptance as a barometer of gastrointestinal and systemic health[327,328]. Elevations in the Firmicutes:Bacteroidetes ratio have been associated with a high-fat diet and obesity[274,328,329], irritable bowel syndrome[330], hypertension[331], type 1 diabetes[276], and HIV[332]. Characteristically, dysbiosis of this ratio heralds a shift in microbiota from obligate anaerobes to nitrate-reducing, facultative anaerobes of the Proteobacteria and Firmicutes phyla[333–335]. The typically low-abundance Enterobacteraceae expand in concert with inflammation[336] and degree of dysbiosis of the Firmicutes:Bacteroidetes ratio[333,337,338]. This shift is particularly notable in the context of inflammatory bowel disease, where massive blooms of pathogenic, nitrate-reducing Salmonella and Escherichia coli are seen in response to inflammation[333,334,338,339]. Presumably, in the setting of inflammation, excess nitrite and nitrate are formed along with reactive oxygen radicals and nitrogen oxides through NO oxidation, thereby increasing the amount of substrate available for respiratory denitrification and rapid bacterial growth[323,337]. Similarly, this concept has been proposed in pathognomonic populations of Pseudomonas and other Proteobacteria in patients with cystic fibrosis[323,337], suggesting that bacterial nitrate metabolism in the gut is an important selective factor in pathogenic bacterial survival, thereby acting to disrupt the luminal microenvironment and alter the generation of NO and uptake of nitrate and nitrite. This alteration in turn can promote chronic inflammation and contribute to the impaired NO signaling and inflammation-rich intravascular environment that is a hallmark PH.

NO Production

Although the majority of ingested nitrate and nitrite are absorbed into the circulation in the proximal intestine, commensal gut bacteria are able to directly produce NO and also modify NO production, potentially influencing regional blood flow, mucosal integrity, microbial diversity, and effective nitrate and nitrite uptake. Many bacteria are capable of reducing nitrate to nitrite to NO via the denitrification pathway. For example, both Lactobacilli and Bifidobacteria have been shown to generate NO from nitrite in culture, while Lactobacilli, Escherichia coli and Salmonella typhimurium all increase cecal NO production when supplied with nitrate[217,233,271,278].

Additionally, some bacteria, such as Lactobaciilus[340] and Streptomyces[341], produce NO via a bacterial NOS oxidation of L-arginine, akin to mammalian endothelial NOS function[342]. Both commensal and pathogenic bacteria possess this capability, although it remains unclear how active these mechanisms are in vivo and exactly what benefit this provides to bacteria. From the standpoint of NO signaling and PH, respiratory denitrification of nitrite and NOS-derived NO likely serve beneficial roles in modifying the local microenvironment, and maintaining adequate blood flow and mucus generation. Alternatively, the intriguing hypothesis has been raised that NO formation may relate to pathogen virulence via biosynthesis of toxins, protection from host oxidative damage, or even stimulation of blood vessel formation in tissue that is beneficial to bacterial proliferation[342].

Another avenue for microbial influence on NO production in the gut is via modulation of hydrogen sulfide (H₂S) metabolism. H₂S is a biologically important thiol that shares significant overlapping signaling activity with NO, reacts with NO-derived reactive species and has been implicated in modulating the pathogenesis of several inflammatory bowel diseases[91,343,344]. At least 50% of intestinal H₂S is derived from a set of commensal Proteobacteria, called the sulfate-reducing bacteria (SRB), either by cysteine degradation or a dissimilatory sulfate reduction reaction[345]. H₂S directly interfaces with nitrogen oxide reduction and the generation of intestinal NO by serving as an electron donor during nitrite reduction to NO by nitrite reductase, or by reacting with the nitrogen oxide S-nitrosothiol[91,102,346]. Notably, H₂S is required for both efficient nitrite reduction to NO in the gut and in NO formation from nitrite in hypoxic systemic tissues[343,346]. Additionally, the major SRB families Desulfovibrionaceae (Desulfovibrio, etc.) and Enterobacteriaceae (Enterobacter, Salmonella, Escherichia, Shigella, etc.), found to be enriched in inflammatory bowel conditions as well as periodontal disease and metabolic syndrome[344,345], overlap significantly with those identified as pathogenic, nitratereducing Proteobacteria in the inflammatory gut and cystic fibrosis lung[323,337]. Indeed, there remains conflicting evidence regarding the precise role of H₂S in the gut[344,347]. On one hand, it appears that lower concentrations of endogenously derived H₂S exert beneficial cytoprotective, antioxidant and anti-inflammatory actions[344,348–351], while on the other, increased H₂S formation by SRB can promote oxidant formation, impair colonic epithelial H₂S detoxification and induce cytotoxicity and disruption of intestinal barrier integrity, thereby driving local and systemic inflammation[344,347,352,353]. Certainly, further understanding of the roles that H₂S concentration, substrate, and location play in inflammation and nitrate metabolism remain to be more fully elucidated. However, intestinal bacterial metabolism of H₂S may represent an additional therapeutic target to modify the bioavailability of nitrite, NO and nitrogen oxides, as well as enteric and systemic inflammation, and potentially vascular injury and PH.

Therapeutic Strategies to Target the Enterosalivary Pathway: Beyond Nitrate Supplementation

With the advent of scientific interest in the microbiome, modulation of the microbiome by prebiotics, probiotics, and antibiotics have gained recent attention as potentially effective avenues for treating a variety of conditions[354–356]. Each strategy seeks to select for a targeted, healthy set of microbiota. Prebiotics, for instance, refers to the use of foods, drugs, or supplements to selectively promote the growth of desired microbiota (Table 1)[357]. Probiotics, refers to the ingestion of live bacteria or bacterial communities in order to recolonize with or select for desired microbiota, while antibiotics refers to the use of anti-microbial drugs to kill unhealthy, or pathogenic bacteria (Table 1)[357]. Many of these targeted strategies have great potential to transition from epidemiological or homeopathic associations to rigorously studied therapeutics for PH and other vascular diseases.

Table 1.

Classifications of potential therapeutic strategies to modulate NO signaling of bacterial nitrate reduction to nitrite, NO and nitrogen oxides in vascular disease and pulmonary hypertension

Prebiotics
Foods, drugs, or food ingredients (supplements) indigestible by the host that selectively enhance the growth and activity of beneficial bacteria or bacterial communities in the gut[357,358].
Diet-based	Supplements
◦Nitrate-rich fruits & vegetables; DASH[161,361,368,369] ◦Vegetarian[359,441] ◦Unsaturated fatty-acids; Mediterranean[362,375]	◦Nitrate/nitrite; beetroot[374] ◦Nitro-fatty acids; S-nitrosothiols[93,442] ◦Unsaturated fatty acids; oleic, linoleic acid[94,105] ◦Conjugated fatty acids; cLA[94,443] ◦Polyphenols[19,444]
Probiotics
Live bacteria or polymicrobial cultures that confer benefit on the host by colonizing or otherwise altering existing bacterial populations and their metabolism[357,400,402].
Strain-specific, single & consortia	Non-specific
◦Bifidobacterium spp.[401,411] ◦Lactobacillus spp.[74,217,279]	◦Fermented food products; yogurt, kombucha[398,400,414] ◦Human samples (e.g. FMT)[445]

Antibiotics & Antimicrobial Agents
Bactericidal drugs or substances that selectively and non-selectively kill or deplete populations of pathogenic bacteria[357].
Non-selective	Broad-spectrum	Targeted
◦Bactericidal mouthwash; chlorhexidine[99,446] ◦Biofilm dispersal; NO, nitrite[235–237]	◦‘Traditional’ antibiotic drugs[421]	◦Bacteriophages[437] ◦Antibiotic-Ab conjugates[436] ◦Antimicrobial peptides[439]

Abbreviations: DASH, Dietary Approaches to Stop Hypertension; cLA, conjugated linoleic acid; FMT, fecal microbial transplantation; NO, nitric oxide; Ab, antibody

Prebiotic therapy: Dietary Interventions

Attempts to define prebiotics as more than just ‘food’ have led to the definition as, “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon”[358]. However, changing one’s diet remains one of the most ubiquitous methods of risk reduction for disease and has potential to alter the microbiome and NO formation in a variety of ways. All dietary manipulations seek to alter systemic metabolic function at some level, though almost no mechanisms of observed effects are completely understood. The current trend in ‘biotic’ therapies is shifting from more generalized dietary recommendations toward more targeted supplementation with or elimination of specific elements of a given diet. Numerous studies have now begun to link dietary features (such as high fat intake, vegetarianism, etc.) and their associated health consequences with changes in the gut microbiome[157,359,360]. Here, the potential relationship between two major dietary strategies, the high fruit and vegetable diet recommended by the Dietary Approaches to Stop Hypertension (DASH) study[361] and the traditional, fatty acid-rich Mediterranean diet[362], and their impact on the enterosalivary nitrate pathway and development of PH and vascular diseases are discussed.

Major epidemiological studies have consistently identified that vegetable- and fruit-rich diets improve every major risk factor for PH and CVD, including blood pressure[361,363,364], ischemic stroke[365], ischemic heart disease[366], obesity, insulin resistance and diabetes[367], and metabolic syndrome[365,366,368,369].

Specifically, green leafy vegetables appear to be most important in conferring these benefits[369]. Green leafy vegetables as well as root vegetables such as beets are known to contain the highest amounts of inorganic nitrate[36,161,370]. In Western countries, a sub-analysis of the DASH study found that nitrate-mediated blood pressure effects were first seen at daily levels of about 160mg of vegetable-based nitrate[361,371], with a typical DASH-compliant diet estimated to contain a range of nitrate from 174–1222mg/day[161]. Unsurprisingly, estimates of average daily nitrate consumption in the U.S. and Europe, regions with high rates of CVD and vascular disease, reach only 40–100mg and 30–180mg, respectively[157,161,372]. Comparing this level of consumption to a study of the high vegetable-containing, traditional Japanese diet, a population with historically low rates of cardiovascular disease, Sobko et al. found the average daily nitrate consumption in Japan to be >1100mg/day for a 60kg person, and associated with increased circulating nitrate and nitrite and with reduced blood pressure[373]. Combining these dietary findings with the numerous studies already reviewed above that demonstrate physiological blood pressure and vascular effects and increased circulating levels of nitrate and nitrite with natural sources of nitrate in beetroot juice[374] and spinach[111], there is compelling evidence that at least some of the beneficial effects of major dietary initiatives are driven by the enterosalivary nitrate pathway.

A second potentially important dietary target in modifying PH risk is the consumption of “healthy” fats in the form of unsaturated fatty acids, such as oleic and linoleic acid[375]. Current dietary investigations have found these fatty acids and dietary nitrate to be concentrated in the traditional Mediterranean diet, a diet with beneficial effects on the development of CVD and its risk factors[362,375–377]. In contrast to the high-nitrate diet of Japan, the Seven Countries Study[378] found lower rates of CVD and cancer with the traditional Mediterranean diet, despite 37% of total daily energy coming from fat, versus only 11% in the traditional Japanese diet[378,379]. Further work has since identified associations in both diets with high content of alpha linoleic acids (ALA), estimated to make up approximately 24% of total energy intake along with oleic acid in the traditional Mediterranean diet[379–381]. Comparatively, U.S. consumption of total polyunsaturated fatty acids and ALA are around 6–7% and 0.6–0.7% of total energy intake, respectively[382,383]. These numbers, along with the relative incidence of CVD and vascular risk factors support the hypothesis of a possible synergistic, diet-mediated NO-effect between inorganic nitrates and unsaturated fatty acids[19]. The specifics of these dietary interventions remain largely speculative and have not been investigated in PH, but could offer a novel strategy for implementation and study.

Mechanistically, synergy between unsaturated fatty acids and ingested nitrate and nitrite may be mediated by the anti-inflammatory effects of electrophilic (electron pair acceptor via Michael addition), nitro-fatty acid (NO₂-FA) formation in the stomach. Under low pH, non-enzymatic reduction of nitrite to NO produces an intermediate nitrogen dioxide radical that can react with dietary, unsaturated, electrophilic fatty acids such as the essential FAs, oleic and linoleic acid, to form nitro-conjugated fatty acids and S-nitrosothiol derivatives[93,384]. NO₂-FAs exert pleiotropic post-translational, anti-oxidant and anti-inflammatory effects[105,321,385] that include inhibition of vascular smooth muscle proliferation and neointima formation after injury, cardioprotection, improved endothelial function[94,386–390], and activation of downstream NO signaling pathways[385,389,391–394]. In a high-fat diet model of PH, Kelley et al. found that mice supplemented with the NO₂-FA, nitro-octadecenoic acid, for 6.5 out of 20 weeks on a high-fat diet had reductions in markers of oxidative stress, inflammation, right ventricular remodeling, and degree of pulmonary pressure elevation seen in mice without NO₂-FA supplementation on the same high-fat diet[391]. Further, supplementation with the conjugated, diene-containing FA, conjugated linoleic acid (cLA), has been shown in itself to be beneficial in obesity and insulin sensitivity in humans, exert powerful anti-inflammatory effects in the setting of ischemia-reperfusion[72,105,384,395], and to alter the gut microbiome[396,397]. Given such evidence, it stands to reason that the full physiological impact of the enterosalivary nitrate pathway can involve both direct nitrite/NO-mediated and NO₂-FA-mediated vascular responses. Further study of the dietary treatment and prevention of PH and vascular disease must, therefore, include consideration of both the nitrate-nitrite-NO and the nitrate-nitrite-NO-FA pathways.

Probiotics

As understanding of the nuanced bacterial interrelationships within the microbiome becomes more sophisticated, the ability to modify community structure and function through the selective introduction of beneficial bacteria in the form of probiotics will likely become more widespread. That is not to say that the practice of ingesting fermented foods for health and entertainment has not existed since Neolithic times[398], though it has only been a relatively short period that medical science has given serious consideration of this concept[399–401]. Formally, the modern definition of probiotics was set in 2001 as consisting of “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”[400,402]. In subsequent years, there has been an explosion of work demonstrating clinical benefit of probiotics in modifying cardiovascular risk, inflammatory and infectious bowel disorders, and even ventilator-associated pneumonia (reviewed in:[399,403]). Yet, the mechanisms of these associations continue to remain unclear and their relationship to clinical outcomes unproven.

The goal of probiotic administration is to functionally alter the microbe-host interaction to favorably affect health. Mechanistically, there exists numerous potential avenues to accomplish this goal, including positively or negatively affecting indigenous microbial populations, integration of probiotic microbes into existing communities, altering microbial and/or host metabolism and gene expression, and indirect perturbations to the ambient microenvironment[400]. While human and animal studies have established the ability to successfully populate the oral cavity or gastrointestinal tract with specific strains, combinations of bacteria, or bacteria genetically modified to produce enzymes or metabolites[74,404–407], it is by no means clear that repopulation by probiotic bacteria must occur to achieve its intended effects[309,407]. Given, however, that probiotic therapy exerts a beneficial effect on dental caries, periodontitis, and pharyngitis in the oral cavity[408–411], as well as on obesity, hypertension, insulin resistance, and heart failure via the gut(reviewed:[399,403]), there is clear potential to treat PH with microbial interventions by modifying either oral or gastrointestinal bacteria.

The overwhelming majority of research into probiotics has focused on the lactic acid fermenting bacteria, Bifidobacterium and Lactobacillus, likely due to the widespread historical reliance on fermented dairy products, consistency in manufacturing, and overall safety[399,401]. Indeed, members of these genera are among the only probiotics accepted in Canada and Italy as ones with sufficient evidence to market beneficial health claims[400] and achieve GRAS status (“generally recognized as safe”) in the U.S.[399]. Lactobacillus spp., in particular, appears to offer an enticing target for modulation of NO-effects. At the genus level, Lactobacilli are common and robust bacteria in the mouth and gut, able to tolerate low pH and to generate NO in culture[74,412]. In polymicrobial environments, Lactobacilli engage in advantageous, mutualistic interactions with other bacteria to survive under hypoxia and alter NO generation[217,413], as well as improve inflammation associated with inflammatory bowel conditions and reduce blood pressure in pre-hypertensive and hypertensive patients[401,403,414].

In particular, the commonly available probiotic strain, L. rhamnosus, is capable of reducing nitrate to nitrite[217], producing NO in the gut in response to oral nitrate administration[74,217,278], and to generate and isomerize cLA and exert anti-obesity effects in diet-induced obese mice in a cLA-dependent manner[279,403,415,416]. Functionally, L. rhamnosus mediates physiological effects in the mouth and has good adherence to saliva-coated surfaces[408,417]. It has also been linked to weight reduction, prevention of ischemia-reperfusion injury, and attenuation of cardiac hypertrophy and post-infarction heart failure in rodents[403,418,419], and beneficial effects in obesity and metabolic syndrome in humans[405,420]. Based on these findings, it is certainly plausible that L. rhamnosus or other probiotic bacterial strains, alone or in combination, may be able to improve nitrate and nitrite signaling in both the oral cavity and the gut, thus potentially influencing PH.

Further work on probiotic strains remains promising in the treatment and prevention of PH. Future studies will need not only to identify and isolate new candidate species, but also explore targeted engineering of beneficial strains to enhance their clinical effects.

Antibiotic & Antimicrobial Agents

In current use, antibiotics are blunt tools of mass microbial destruction that can permanently alter the microbiome and predispose to numerous diseases such as obesity and diabetes[421]. Overuse and antibiotic resistance have proliferated[422] to the point that the U.S. Food and Drug Administration has now banned the public sale of certain antibacterial soaps[423]. Recent ecological findings show that even low levels of common antibiotics contaminate groundwater and soil and can disrupt bacterial denitrification[424–426], possibly exerting pressure on nitrate- and NO-signaling through agriculture, human food sources, and even disease pathology. While broad discussion of antibiotics is far beyond the scope of this review, two very speculative concepts related to nitrate-signaling and PH will be briefly mentioned.

First, the logical concern resulting from the finding that vascular health may depend on the oral microbiome is to ask if current oral hygiene practices, particularly the use of mouthwash, could be causing more harm than good. After publication by Ahluwalia et al. that 7 days of chlorhexidine mouthwash significantly raises resting blood pressures in healthy volunteers[106], such questions became the subject of widespread public curiosity[427]. However, the answer remains unclear. Two studies have since shown increased blood pressures after chlorhexidine-containing mouthwash treatment in a population of hypertensive men on antihypertensive therapy[428] and during exercise in healthy volunteers[429], while a recent crossover study in healthy women showed no blood pressure effects with similar treatment[430]. Conversely, epidemiological studies consistently show that in the general population, good oral hygiene (specifically frequent tooth brushing and mouthwash use) protects against development of hypertension and CVD[431–433]. Such incongruities may be in part explained by evidence suggesting that one’s choice in mouthwash (from the strongest bactericidal formulations with chlorhexidine, to weaker ones such as with triclosan or cetylpyridinium, to the widely available and weakest antiseptic formulations) greatly impacts the degree of bacterial nitrate reduction and blood pressure effects[99,429,434], and that brushing with even bactericidal toothpaste exerts little to no effect on bacterial nitrate reduction[434]. Clearly, further targeted studies will be needed to fully understand if regular antimicrobial mouthwash use truly impacts the risk for development of PH and its risk factors.

Second, the ability to design and deliver targeted, bacteria- or community-specific antimicrobial therapies to selectively disrupt or augment oral and gut microbial populations and their nitrate and NO metabolism is a very real future[435]. Nitrite, for instance, disrupts protective biofilm formation by pathogenic bacteria and is currently under investigation as an adjunct to standard antibiotic therapy[235,236]. Potentially, a combination of nitrite-mediated biofilm dispersion and use of an antibiotic-conjugated antibody[436] or bacteriophage[437] with species-selectively may offer a multifaceted approach to target antimicrobial populations in the mouth. Yet, it is the discovery of bacteria-produced, antimicrobial peptides by human commensal bacteria that offer potentially significant informational and therapeutic value[438]. Recently, a Streptococcus mutans-specific peptide was developed and tested in a model of human saliva, where selective depletion of S. mutans triggered community-wide shifts in major nitrate-reducing and non-nitrate-reducing populations[439]. The implications of such a tool could allow greater understanding of bacterial networking and their metabolic interdependencies as well as the development of new tools to study and modify their nitrate-reducing activity.

Conclusions

The breadth of signaling responses instigated by nitrate, nitrite, NO and downstream products of nitrogen oxide reactions uniquely exists at the nexus of NO biology and the human-microbe interface. It is clear that these reactions are highly dependent on, and can be readily altered by, oral and gut bacteria separately or in combination. Whether changes to resident microbiota are beneficial or harmful in PH is unknown, but they could influence response to drug therapy, and augmentation of key bacteria or bacterial functions may improve efficacy of therapies with fewer side effects. There is obvious potential for the development of nitrate- and nitrite-based therapies for cardiovascular disease, PH, and their risk factors, and such studies are currently ongoing. Relationships of the microbiome to PH have never been directly examined, but represent a novel testable and modifiable factor in disease pathogenesis.

Future directions will need to include mechanistic and direct clinical testing of both oral nitrate therapy and microbial-based therapeutics or probiotics in PH. Further work is needed to understand the systemic role that enterosalivary nitrate circulation plays in pulmonary vascular health, onset of disease and its progression. The safety of brief and chronic administration of supplemental nitrate and nitrite in the context of PH and dysfunctional NO signaling must be ascertained[440], as well as the potential to generate formulations that are more bioavailable and efficacious while maintaining the relative paucity of negative effects. From the standpoint of microbial nitrate reduction, deeper insights must be ascertained into the complex relationships between bacteria and their local community, their host, and other factors such as fungi, viruses, and bacteriophages.

Despite these complexities, a rich fund of knowledge already exists in the form of effective PH therapies targeting NO signaling and ongoing clinical testing of inhaled and oral formulations of inorganic nitrate and nitrite, as well as synthetic nitro-fatty acids. The study of probiotics has identified specific bacterial strains and their combinations that are capable of inducing pre-clinical and clinical effects. These and future efforts should shed new light on the impact that metabolic convergence of the microbiome and nitrogen oxides will have on the treatment and prevention of PH, CVD, and other vascular diseases.

Highlights.

• The microbiome may influence pulmonary hypertension via nitrogen oxide signaling.

• Oral microbiota are necessary for nitrate reduction to vasoactive nitrogen oxides.

• Dietary nitrate and nitrite are major sources of exogenous nitrogen oxides.

• Dietary nitrate in vegetables exerts pleiotropic, beneficial nitrogen oxide effects.

• Diet and probiotic therapy can alter beneficial nitrate and fatty acid metabolism.