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Having high blood pressure puts you at risk for heart disease and stroke, which are leading causes of death in the USA and worldwide. One out of every three 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 reduces 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 antiseptic mouthwash or overuse 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.

The microbiota of the human lower intestinal tract helps maintain healthy host physiology, for example through nutrient acquisition and bile acid recycling, but specific positive contributions of the oral microbiota to host health are not well established. Nitric oxide (NO) homeostasis is crucial to mammalian physiology. The recently described entero-salivary nitrate-nitrite-nitric oxide pathway has been shown to provide bioactive NO from dietary nitrate sources. Interestingly, this pathway is dependent upon oral nitrate-reducing bacteria, since humans lack this enzyme activity. This pathway appears to represent a newly recognized symbiosis between oral nitrate-reducing bacteria and their human hosts in which the bacteria provide nitrite and nitric oxide from nitrate reduction. Here we measure the nitrate-reducing capacity of tongue-scraping samples from six healthy human volunteers, and analyze metagenomes of the bacterial communities to identify bacteria contributing to nitrate reduction. We identified 14 candidate species, seven of which were not previously believed to contribute to nitrate reduction. We cultivated isolates of four candidate species in single- and mixed-species biofilms, revealing that they have substantial nitrate- and nitrite-reduction capabilities. Colonization by specific oral bacteria may thus contribute to host NO homeostasis by providing nitrite and nitric oxide. Conversely, the lack of specific nitrate-reducing communities may disrupt the nitrate-nitrite-nitric oxide pathway and lead to a state of NO insufficiency. These findings may also provide mechanistic evidence for the oral systemic link. Our results provide a possible new therapeutic target and paradigm for NO restoration in humans by specific oral bacteria.

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Nitrite has emerged as an endogenous signaling molecule with potential therapeutic implications for cardiovascular disease. Steady-state levels of nitrite are derived in part from dietary sources; therefore, we investigated the effects of dietary nitrite and nitrate supplementation and deficiency on NO homeostasis and on the severity of myocardial ischemia-reperfusion (MI/R) injury. Mice fed a standard diet with supplementation of nitrite (50 mg/liter) in their drinking water for 7 days exhibited significantly higher plasma levels of nitrite, exhibited significantly higher myocardial levels of nitrite, nitroso, and nitrosyl-heme, and displayed a 48% reduction in infarct size (Inf) after MI/R. Supplemental nitrate (1 g/liter) in the drinking water for 7 days also increased blood and tissue NO products and significantly reduced Inf. A time course of ischemia-reperfusion revealed that nitrite was consumed during the ischemic phase, with an increase in nitroso/nitrosyl products in the heart. Mice fed a diet deficient in nitrite and nitrate for 7 days exhibited significantly diminished plasma and heart levels of nitrite and NO metabolites and a 59% increase in Inf after MI/R. Supplementation of nitrite in the drinking water for 7 days reversed the effects of nitrite deficiency. These data demonstrate the significant influence of dietary nitrite and nitrate intake on the maintenance of steady-state tissue nitrite/nitroso levels and illustrate the consequences of nitrite deficiency on the pathophysiology of MI/R injury. Therefore, nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a treatment modality for cardiovascular disease.

The oral microbiome has the potential to provide an important symbiotic function in human blood pressure physiology by contributing to the generation of nitric oxide (NO), an essential cardiovascular signaling molecule. NO is produced by the human body via conversion of arginine to NO by endogenous nitric oxide synthase (eNOS) but eNOS activity varies by subject. Oral microbial communities are proposed to supplement host NO production by reducing dietary nitrate to nitrite via bacterial nitrate reductases. Unreduced dietary nitrate is delivered to the oral cavity in saliva, a physiological process termed the enterosalivary circulation of nitrate. Previous studies demonstrated that disruption of enterosalivary circulation via use of oral antiseptics resulted in increases in systolic blood pressure. These previous studies did not include detailed information on the oral health of enrolled subjects. Using 16S rRNA gene sequencing and analysis, we determined whether introduction of chlorhexidine antiseptic mouthwash for 1 week was associated with changes in tongue bacterial communities and resting systolic blood pressure in healthy normotensive individuals with documented oral hygiene behaviors and free of oral disease. Tongue cleaning frequency was a predictor of chlorhexidine-induced changes in systolic blood pressure and tongue microbiome composition. Twice-daily chlorhexidine usage was associated with a significant increase in systolic blood pressure after 1 week of use and recovery from use resulted in an enrichment in nitrate-reducing bacteria on the tongue. Individuals with relatively high levels of bacterial nitrite reductases had lower resting systolic blood pressure. These results further support the concept of a symbiotic oral microbiome contributing to human health via the enterosalivary nitrate-nitrite-NO pathway. These data suggest that management of the tongue microbiome by regular cleaning together with adequate dietary intake of nitrate provide an opportunity for the improvement of resting systolic blood pressure.

Mammalian tissues produce nitric oxide (NO) to modify proteins at heme and sulfhydryl sites, thereby regulating vital cell functions. The majority of NO produced is widely assumed to be neutralized into supposedly inert oxidation products including nitrite (NO 2 − ). Here we show that nitrite, also ubiquitous in dietary sources, is remarkably efficient at modifying the same protein sites, and that physiological nitrite concentrations account for the basal levels of these modifications in vivo . We further find that nitrite readily affects cyclic GMP production, cytochrome P 450 activities, and heat shock protein 70 and heme oxygenase-1 expression in a variety of tissues. These cellular activities of nitrite, combined with its stability and abundance in vivo , suggest that this anion has a distinct and important signaling role in mammalian biology, perhaps by serving as an endocrine messenger and synchronizing agent. Thus, nitrite homeostasis may be of great importance to NO biology.

Endothelial NOS (eNOS)-derived NO has long been considered a paracrine signaling molecule only capable of affecting nearby cells because of its short half-life in blood and relatively limited diffusion distance in tissues. To date, no studies have demonstrated that endogenously generated NO possesses a clearly defined endocrine function. Therefore, we evaluated whether enzymatic generation of NO in the heart is capable of modulating remote physiological actions and cell signaling. Mice with cardiac-specific overexpression of the human eNOS gene (CS-eNOS-Tg) were used to address this hypothesis. Cardiac-specific eNOS overexpression resulted in significant increases in nitrite, nitrate, and nitrosothiols in the heart, plasma, and liver. To examine whether the increase in hepatic NO metabolites could modulate cytoprotection, we subjected CS-eNOS-Tg mice to hepatic ischemia-reperfusion (I/R) injury. CS-eNOS-Tg mice displayed a significant reduction in hepatic I/R injury (4.2-fold reduction in the aminotransferase and a 3.5-fold reduction in aspartate aminotransferase) compared with WT littermates. These findings demonstrate that endogenously derived NO is transported in the blood, metabolized in remote organs, and mediates cytoprotection in the setting of I/R injury. This study presents clear evidence for an endocrine role of NO generated endogenously from eNOS and provides additional evidence for the profound cytoprotective actions of NO in the setting of I/R injury.

There is mounting evidence that the established paradigm of nitric oxide (NO) biochemistry, from formation through NO synthases, over interaction with soluble guanylyl cyclase, to eventual disposal as nitrite/nitrate, represents only part of a richer chemistry through which NO elicits biological signaling. Additional pathways have been suggested that include interaction of NO-derived metabolites with thiols and metals to form S-nitrosothiols (RSNOs) and metal nitrosyls. Despite the overwhelming attention paid in this regard to RSNOs, little is known about the stability of these species, their significance outside the circulation, and whether other nitros(yl)ation products are of equal importance. We here show that N-nitrosation and heme-nitrosylation are indeed as ubiquitous as S-nitrosation in vivo and that the products of these reactions are constitutively present throughout the organ system. Our study further reveals that all NO-derived products are highly dynamic, have fairly short lifetimes, and are linked to tissue oxygenation and redox state. Experimental evidence further suggests that nitroso formation occurs substantially by means of oxidative nitrosylation rather than NO autoxidation, explaining why S-nitrosation can compete effectively with nitrosylation. Moreover, tissue nitrite can serve as a significant extravascular pool of NO during brief periods of hypoxia, and tissue nitrate/nitrite ratios can serve as indicators of the balance between local oxidative and nitrosative stress. These findings vastly expand our understanding of the fate of NO in vivo and provide a framework for further exploration of the significance of nitrosative events in redox sensing and signaling. The findings also raise the intriguing possibility that N-nitrosation is directly involved in the modulation of protein function.

Nitric oxide (NO) is involved in number of physiological and pathological events. Our previous studies demonstrated a differential expression of NO signaling components in mouse and human ES cells. Here, we demonstrate the effect of NO donors and soluble guanylyl cyclase (sGC) activators in differentiation of ES cells into myocardial cells. Our results with mouse and human ES cells demonstrate an increase in Nkx2.5 and myosin light chain (MLC2) mRNA expression on exposure of cells to NO donors and a decrease in mRNA expression of both cardiac-specific genes with nonspecific NOS inhibitor and a concomitant increase and decrease in the mRNA levels of sGC α₁ subunit. Although sGC activators alone exhibited an increase in mRNA expression of cardiac genes (MLC2 and Nkx2.5), robust inductions of mRNA and protein expression of marker genes were observed when NO donors and sGC activators were combined. Measurement of NO metabolites revealed an increase in the nitrite levels in the conditioned media and cell lysates on exposure of cells to the different concentrations of NO donors. cGMP analysis in undifferentiated stem cells revealed a lack of stimulation with NO donors. Differentiated cells however, acquired the ability to be stimulated by NO donors. Although, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridine (BAY 41-2272) alone was able to stimulate cGMP accumulation, the combination of NO donors and BAY 41-2272 stimulated cGMP levels more than either of the agents separately. These studies demonstrate that cGMP-mediated NO signaling plays an important role in the differentiation of ES cells into myocardial cells.

Endothelial NO production results in local formation of adducts that may act as storage forms of NO. Because little is known about their chemical nature, concentrations, and possible role in vascular biology, we sought to characterize those species basally present in rat aorta, using two independent approaches. In the first approach, tissue homogenates were analyzed by using chemiluminescence- and ion-chromatography-based techniques that allow trace-level quantification of NO-related compounds in complex biological matrices. In the second approach, NO stores were characterized by their ability to release NO when illuminated with light and subsequently relax vascular smooth muscle (photorelaxation). The latter included a careful assessment of action spectra for photorelaxation, taking into account the light-scattering properties of the tissue and the storage depletion rates induced by exposure to controlled levels of light. Biochemical analyses revealed that aortic tissues contained 10 ± 1 µM nitrite, 42 ± 7 µM nitrate, 40 ± 6 nM S-nitroso, and 33 ± 6 nM N-nitroso compounds (n = 4-8). The functional data obtained suggest that the NO photolytically released in the tissue originated from species with photophysical properties similar to those reported for low-molecular-weight S-nitrosothiols, as well as from nitrite. The relative contribution of these potential NO stores to the extent of photorelaxation was consistent with their concentrations detected biochemically in vascular tissue when their photoactivity was taken into account. We conclude that intravascular nitroso species and nitrite both have the potential to release physiologically relevant quantities of NO independent of enzymatic control by NO synthase.