Supplemental nitrite increases choroidal neovascularization in mice.

Low doses of nitrite, close to physiological levels, increase blood flow in normal and ischemic tissues through a nitric oxide (NO) dependent mechanism. Given that nitrite therapy and dietary supplementation with vegetables high in nitrate (e.g. beets) are gaining popularity we decided to determine if low doses of nitrite impact the development of choroidal neovascularization (CNV), a key feature of wet age related macular degeneration (AMD). Sodium nitrite (at 50 mg/L, 150 mg/L, and 300 mg/L), nitrate (1 g/L) or water alone were provided in the drinking water of C57BL/6 J mice aged 2 or 12 months. Mice were allowed to drink ad libitum for 1 week at which time laser-induced choroidal neovascularization (L-CNV) was induced. The mice continued to drink the supplemented water ad libitum for a further 14 days at which point optical coherence tomography (OCT) was performed to determine the volume of the CNV lesion. Blood was drawn to determine nitrite and nitrate levels and eyes taken for histology. CNV volume was 2.86 × 107 µm3 (±0.4 × 107) in young mice on water alone but CNV volume more than doubled to >6.9 × 107 µm3 (±0.8 × 107) in mice receiving 300 mg/L nitrite or 7.34 × 107 µm3 (±1.4 × 107) in 1 g/L nitrate (p < 0.01). A similar trend was observed in older mice. CNV volume was 5.3 × 107 µm3 (±0.5 × 107) in older mice on water alone but CNV volume almost doubled to approximately 9.3 × 107 µm3 (±1.1 × 107) in mice receiving 300 mg/L nitrite or 8.7 × 107 µm3 (±0.9 × 107) 1 g/L nitrate (p < 0.01). Plasma nitrite levels were highest in young mice receiving 150 mg/L in the drinking water with no changes in plasma nitrate observed. In older mice, drinking water nitrite did not significantly change plasma nitrite, but plasma nitrate was increased. Plasma nitrate was elevated in both young and old mice provided with nitrate supplemented drinking water. Our data demonstrate that the CNV lesion is larger in older mice compared to young and that therapeutic levels of oral nitrite increase the volume of CNV lesions in both young and older mice. Therapeutic nitrite or nitrate supplementation should be used with caution in the elderly population prone to CNV.

Bronchopulmonary dysplasia is associated with reduced oral nitrate reductase activity in extremely preterm infants.

Oral microbiome mediated nitrate reductase (NR) activity regulates nitric oxide (NO) bioavailability and signaling. While deficits in NO-bioavailability impact several morbidities of extreme prematurity including bronchopulmonary dysplasia (BPD), whether oral NR activity is associated with morbidities of prematurity is not known. We characterized NR activity in extremely preterm infants from birth until 34 weeks' post menstrual age (PMA), determined whether changes in the oral microbiome contribute to changes in NR activity, and determined whether changes in NR activity correlated with disease. In this single center prospective cohort study (n = 28), we observed two surprising findings: (1) NR activity unexpectedly peaked at 29 weeks' PMA (p < 0.05) and (2) when infants were stratified for BPD status, infants who developed BPD had significantly less NR activity at 29 weeks' PMA compared to infants who did not develop BPD. Oral microbiota and NR activity may play a role in BPD development in extremely preterm infants, indicating potential for disease prediction and therapeutic targeting.

Measuring nitrate reductase activity from human and rodent tongues.

Reduction of salivary nitrate to nitrite by oral microbes expressing nitrate-reductase has emerged as a crucial pathway in systemic NO homeostasis in humans and other mammals. Selective depletion of oral microbes prevents dietary nitrate-dependent lowering of blood pressure, inhibition of platelet aggregation and ischemic injury. To date, most studies interrogate enterosalivary nitrate reduction by following changes in saliva or plasma nitrite and NO-signaling (functional) end points. Little is known about whether, and if so how, nitrate-reductase enzymatic activity per se (i.e. independent of nitrate levels) is a variable and may account for any individual to individual variation. Here, we describe a minimally invasive protocol that allows for NR activity determination from human, rat and mouse tongue scrapes/swabs. We validate this method using selective application of antiseptic agents to the distal tongue surface which decreased NR activity by >80% and show that bacterial number is a significant variable in measured NR activities between males and females. Also, we show that NR activity is >80% lower in smokers (humans) and after bromine gas exposure (mice), suggesting that exposure to inhaled reactive substances inhibit NR activity identifying a potentially new mechanism by which environmental toxicants promote dysfunction in NO-bioavailability. The described method will facilitate studies testing whether NR specific activity is a variable in different pathophysiologic settings, and in turn how this activity modulates enterosalivary nitrate-reduction.

Nitric oxide produced in Peyer's patches exhibits antiapoptotic activity contributing to an antimicrobial effect in murine salmonellosis.

Salmonella species normally infect hosts via the oral-fecal route. We previously reported that NO had potent host defense functions in murine salmonellosis, not only via a direct antibacterial effect but also because it was cytoprotective for infected host cells. Here, we used an oral route to infect iNOS-deficient mice infected with S. enterica serovar Typhimurium to further investigate the cytoprotective role of NO in preventing damage caused by Salmonella organisms in PP. Oral bacterial challenge (2 x 10(5) CFU, or >100 LD(50)) produced a more severe infection and greater lethality in iNOS-deficient mice than in iNOS-competent mice. We used specific antibodies to S. enterica Typhimurium, neutrophils, iNOS, nitrotyrosine, and dendritic cells (CD11c-positive) in histochemical and immunohistochemical studies to examine infected PP tissues. S. enterica Typhimurium colonization in PP from iNOS-deficient mice was significantly higher than that in wild-type mice. Histochemical assays showed extensive cellular damage in PP. We then examined PP tissues for apoptosis by means of in situ TUNEL analysis and by measuring caspase-3 specific activity in tissue homogenates. Increased numbers of TUNEL-positive cells and severe granulomatous inflammation with increased infiltration of neutrophils and macrophages were observed during infection in iNOS-deficient mice compared with wild-type mice. iNOS-deficient mice had increased numbers of dendritic cells and significantly higher caspase-3-specific activity in PP. These data confirm that NO exerts its protective function not only through direct antibacterial action, but also by preventing apoptosis and thereby contributing to antimicrobial defense during salmonellosis.