Long-term dietary nitrite and nitrate deficiency causes the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice.

AIMS/HYPOTHESIS: Nitric oxide (NO) is synthesised not only from L-arginine by NO synthases (NOSs), but also from its inert metabolites, nitrite and nitrate. Green leafy vegetables are abundant in nitrate, but whether or not a deficiency in dietary nitrite/nitrate spontaneously causes disease remains to be clarified. In this study, we tested our hypothesis that long-term dietary nitrite/nitrate deficiency would induce the metabolic syndrome in mice. METHODS: To this end, we prepared a low-nitrite/nitrate diet (LND) consisting of an amino acid-based low-nitrite/nitrate chow, in which the contents of L-arginine, fat, carbohydrates, protein and energy were identical with a regular chow, and potable ultrapure water. Nitrite and nitrate were undetectable in both the chow and the water. RESULTS: Three months of the LND did not affect food or water intake in wild-type C57BL/6J mice compared with a regular diet (RD). However, in comparison with the RD, 3 months of the LND significantly elicited visceral adiposity, dyslipidaemia and glucose intolerance. Eighteen months of the LND significantly provoked increased body weight, hypertension, insulin resistance and impaired endothelium-dependent relaxations to acetylcholine, while 22 months of the LND significantly led to death mainly due to cardiovascular disease, including acute myocardial infarction. These abnormalities were reversed by simultaneous treatment with sodium nitrate, and were significantly associated with endothelial NOS downregulation, adiponectin insufficiency and dysbiosis of the gut microbiota. CONCLUSIONS/INTERPRETATION: These results provide the first evidence that long-term dietary nitrite/nitrate deficiency gives rise to the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice, indicating a novel pathogenetic role of the exogenous NO production system in the metabolic syndrome and its vascular complications.

Effect of caffeine contained in a cup of coffee on microvascular function in healthy subjects.

Recent epidemiological studies have demonstrated that coffee drinking is associated with reduced mortality of cardiovascular disease. However, its precise mechanisms remain to be clarified. In this study, we examined whether single ingestion of caffeine contained in a cup of coffee improves microvascular function in healthy subjects. A double-blind, placebo-controlled, crossover study was performed in 27 healthy volunteers. A cup of either caffeinated or decaffeinated coffee was drunk by the subjects, and reactive hyperemia of finger blood flow was assessed by laser Doppler flowmetry. In an interval of more than 2 days, the same experimental protocol was repeated with another coffee in a crossover manner. Caffeinated coffee intake slightly but significantly elevated blood pressure and decreased finger blood flow as compared with decaffeinated coffee intake. There was no significant difference in heart rate between caffeinated and decaffeinated coffee intake. Importantly, caffeinated coffee intake significantly enhanced post-occlusive reactive hyperemia of finger blood flow, an index of microvascular endothelial function, compared with decaffeinated coffee intake. These results provide the first evidence that caffeine contained in a cup of coffee enhances microvascular function in healthy individuals.

Development of an experimentally useful model of acute myocardial infarction: 2/3 nephrectomized triple nitric oxide synthases-deficient mouse.

We investigated the effect of subtotal nephrectomy on the incidence of acute myocardial infarction (AMI) in mice deficient in all three nitric oxide synthases (NOSs). Two-thirds nephrectomy (NX) was performed on male triple NOSs(-/-) mice. The 2/3NX caused sudden cardiac death due to AMI in the triple NOSs(-/-) mice as early as 4months after the surgery. The 2/3NX triple NOSs(-/-) mice exhibited electrocardiographic ST-segment elevation, reduced heart rate variability, echocardiographic regional wall motion abnormality, and accelerated coronary arteriosclerotic lesion formation. Cardiovascular risk factors (hypertension, hypercholesterolemia, and hyperglycemia), an increased number of circulating bone marrow-derived vascular smooth muscle cell (VSMC) progenitor cells (a pro-arteriosclerotic factor), and cardiac up-regulation of stromal cell-derived factor (SDF)-1α (a chemotactic factor of the progenitor cells) were noted in the 2/3NX triple NOSs(-/-) mice and were associated with significant increases in plasma angiotensin II levels (a marker of renin-angiotensin system activation) and urinary 8-isoprostane levels (a marker of oxidative stress). Importantly, combined treatment with a clinical dosage of an angiotensin II type 1 receptor blocker, irbesartan, and a calcium channel antagonist, amlodipine, markedly prevented coronary arteriosclerotic lesion formation and the incidence of AMI and improved the prognosis of those mice, along with ameliorating all those pro-arteriosclerotic parameters. The 2/3NX triple NOSs(-/-) mouse is a new experimentally useful model of AMI. Renin-angiotensin system activation, oxidative stress, cardiovascular risk factors, and SDF-1α-induced recruitment of bone marrow-derived VSMC progenitor cells appear to be involved in the pathogenesis of AMI in this model.

Higher overall survival rates of oral squamous cell carcinoma treated with metronomic neoadjuvant chemotherapy.

Distant metastasis is an important prognostic factor for oral squamous cell carcinoma (OSCC). It involves the direct spread of tumor cells through blood vessels or via lymph nodes; however, there are currently no well-established treatments for its prevention in patients with OSCC. To investigate the impact of metronomic neoadjuvant chemotherapy on OSCC, we conducted a retrospective analysis of the efficacy of neoadjuvant chemotherapy with S-1 alone. Fifty-four patients underwent up-front surgery, while 106 received neoadjuvant chemotherapy with S-1 alone. A serious adverse event occurred in one of patient treated with neoadjuvant chemotherapy (1%); however, all patients underwent resection. The 5-year overall survival rate was higher with S-1 than with up-front surgery (96% vs. 81%, P = 0.002). Moreover, neoadjuvant chemotherapy significantly increased the overall survival rate of patients with poorly or moderately differentiated tumors, but not those with well-differentiated tumors. By analyzing a cohort of 523 head and neck squamous cell carcinoma (HNSCC) patients in the Cancer Genome Atlas, we identified genetic variants associated with histological differentiation. The frequency of pathogenic/likely pathogenic variants or deletions in 5 genes associated with HNSCC correlated with histological differentiation, some of which indicated the activation of the Wnt/ß-catenin pathway in well-differentiated HNSCC. The vessel marker CD31 was highly expressed in poorly differentiated OSCC, whereas the anti-angiogenic molecule, LCN2, which is induced by the activation of the Wnt pathway, was highly expressed in well-differentiated OSCC. The present study showed that overall survival rates were higher in patients with poorly or moderately differentiated OSCC who received metronomic neoadjuvant chemotherapy, which was attributed to a difference in angiogenesis based on the characteristic landscape of pathogenic mutations according to histological differentiation.

Long-term treatment with san'o-shashin-to, a kampo medicine, markedly ameliorates cardiac ischemia-reperfusion injury in ovariectomized rats via the redox-dependent mechanism.

BACKGROUND: Hormone replacement therapy has failed to reduce ischemic cardiovascular events in climacteric women. To explore alternative therapy, we examined whether san'o-shashin-to (TJ-113), a kampo medicine, ameliorates cardiac ischemia-reperfusion (IR) injury in a climacteric rat model. METHODS AND RESULTS: Cardiac function and infarct size after IR were significantly exacerbated in ovariectomized rats as compared with sham-operated rats, whereas long-term treatment with a clinical dosage of TJ-113 for 4 weeks markedly improved these functional and morphological changes. Myocardial inducible nitric oxide synthase (iNOS) expression and peroxynitrite levels were significantly higher in ovariectomized rats compared with sham-operated rats, and long-term TJ-113 treatment significantly reduced these oxidative changes. Furthermore, myocardial manganese superoxide dismutase (Mn-SOD) activity was significantly lower in ovariectomized than in sham-operated rats, and long-term TJ-113 treatment significantly restored antioxidant activity. Importantly, those beneficial actions of TJ-113 were significantly inhibited by the estrogen receptor antagonist, fulvestrant, and the phytoestrogen, emodin, a TJ-113 ingredient, mimicked the actions of TJ-113, suggesting involvement of emodin in the effects of TJ-113. CONCLUSIONS: These results provide the first evidence that long-term treatment with a clinical dosage of TJ-113 markedly ameliorates cardiac IR injury in ovariectomized rats via inhibition of iNOS expression, suppression of peroxynitrite formation, and restoration of Mn-SOD activity. TJ-113 may be a novel therapeutic option in the treatment of ischemic heart disease in climacteric women.

EphA4 signaling is involved in the phenotype of well-differentiated oral squamous cell arcinoma with decreased tumor immunity.

Metronomic chemotherapy is defined as a high-frequency low-dose schedule of chemotherapy drug administration. Although metronomic chemotherapy is widely used, the mechanisms underlying resistance to metronomic chemotherapy remain unclear. Therefore, we herein conducted a single institutional phase I/II trial to assess the efficacy and safety of metronomic chemotherapy with bleomycin plus S-1, an oral 5-FU prodrug, in the neoadjuvant setting for patients with oral squamous cell carcinoma (OSCC). The response rate of well-differentiated OSCC to metronomic chemotherapy was significantly lower. We investigated differences in molecular profiles between poorly or moderately differentiated head and neck squamous cell carcinoma (HNSCC) and well-differentiated HNSCC from patients with HNSCC TCGA data. EphA4 expression positively correlated with histological differentiation. An upstream regulator analysis correlated with EphA4 expression identified pathways associated with decreased mTORC1 signaling and T cell activation, including TCR, CD3, CD28, and CD40LG. An EphA4 blocking peptide (KYL) induced mTOR activation in well-differentiated OSCC cell lines. Plasmacytoid dendritic cell and CD8+ T cell numbers were higher in the microenvironment of poorly or moderately differentiated HNSCC than in that of well-differentiated HNSCC. Well-differentiated HNSCC had the characteristics of "cold tumors" (immune-excluded tumors). Moreover, KYL used with chemotherapeutic drugs synergistically increased cancer cell death. Well-differentiated OSCC is depleted of immune cells, which may be partly explained by the receptor tyrosine kinase EphA4.

[Long-term dietary nitrite and nitrate deficiency causes metabolic syndrome, endothelial dysfunction, and cardiovascular death in mice].

Nitric oxide (NO) is synthesized not only from L-arginine by NO synthases (NOSs), but also from its inert metabolites, nitrite and nitrate. Green leafy vegetables are abundant in nitrate, however whether or not a deficiency in dietary nitrite/nitrate spontaneously causes disease remains to be clarified. In this study, we tested our hypothesis that long-term dietary nitrite/nitrate deficiency induces metabolic syndrome (MetS) in mice. To this end, we prepared a low nitrite/nitrate diet (LND) consisting of an amino acid-based low nitrite/nitrate chow in which the contents of L-arginine, fat, carbohydrates, protein, and energy were identical with a regular chow, and potable ultrapure water. Nitrite and nitrate were undetectable in both the chow and the water. Intriguingly, in comparison with a regular diet, 3 months of the LND significantly elicited visceral adiposity, dyslipidaemia, and glucose intolerance; 18 months of the LND significantly provoked increased body weight, hypertension, insulin resistance, and impaired endothelium-dependent relaxations to acetylcholine; and 22 months of the LND significantly led to death due to cardiovascular disease, including acute myocardial infarction. These abnormalities were reversed by simultaneous treatment with sodium nitrate, and were significantly associated with endothelial NOS down-regulation, adiponectin insufficiency, and gut microbiota dysbiosis. These results provide the first evidence that long-term dietary nitrite/nitrate deficiency gives rise to MetS, endothelial dysfunction, and cardiovascular death in mice, indicating a novel pathogenetic role of the exogenous NO production system in MetS and its vascular complications.