AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis.

Advanced age and unhealthy dietary habits contribute to the increasing incidence of obesity and type 2 diabetes. These metabolic disorders, which are often accompanied by oxidative stress and compromised nitric oxide (NO) signaling, increase the risk of adverse cardiovascular complications and development of fatty liver disease. Here, we investigated the therapeutic effects of dietary nitrate, which is found in high levels in green leafy vegetables, on liver steatosis associated with metabolic syndrome. Dietary nitrate fuels a nitrate-nitrite-NO signaling pathway, which prevented many features of metabolic syndrome and liver steatosis that developed in mice fed a high-fat diet, with or without combination with an inhibitor of NOS (l-NAME). These favorable effects of nitrate were absent in germ-free mice, demonstrating the central importance of host microbiota in bioactivation of nitrate. In a human liver cell line (HepG2) and in a validated hepatic 3D model with primary human hepatocyte spheroids, nitrite treatment reduced the degree of metabolically induced steatosis (i.e., high glucose, insulin, and free fatty acids), as well as drug-induced steatosis (i.e., amiodarone). Mechanistically, the salutary metabolic effects of nitrate and nitrite can be ascribed to nitrite-derived formation of NO species and activation of soluble guanylyl cyclase, where xanthine oxidoreductase is proposed to mediate the reduction of nitrite. Boosting this nitrate-nitrite-NO pathway results in attenuation of NADPH oxidase-derived oxidative stress and stimulation of AMP-activated protein kinase and downstream signaling pathways regulating lipogenesis, fatty acid oxidation, and glucose homeostasis. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against liver steatosis associated with metabolic dysfunction.

Endothelial-Tumor Cell Interaction in Brain and CNS Malignancies.

Glioblastoma and other brain or CNS malignancies (like neuroblastoma and medulloblastoma) are difficult to treat and are characterized by excessive vascularization that favors further tumor growth. Since the mean overall survival of these types of diseases is low, the finding of new therapeutic approaches is imperative. In this review, we discuss the importance of the interaction between the endothelium and the tumor cells in brain and CNS malignancies. The different mechanisms of formation of new vessels that supply the tumor with nutrients are discussed. We also describe how the tumor cells (TC) alter the endothelial cell (EC) physiology in a way that favors tumorigenesis. In particular, mechanisms of EC-TC interaction are described such as (a) communication using secreted growth factors (i.e., VEGF, TGF-ß), (b) intercellular communication through gap junctions (i.e., Cx43), and (c) indirect interaction via intermediate cell types (pericytes, astrocytes, neurons, and immune cells). At the signaling level, we outline the role of important mediators, like the gasotransmitter nitric oxide and different types of reactive oxygen species and the systems producing them. Finally, we briefly discuss the current antiangiogenic therapies used against brain and CNS tumors and the potential of new pharmacological interventions that target the EC-TC interaction.

Control of human energy expenditure by cytochrome c oxidase subunit IV-2.

Resting metabolic rate (RMR) in humans shows pronounced individual variations, but the underlying molecular mechanism remains elusive. Cytochrome c oxidase (COX) plays a key role in control of metabolic rate, and recent studies of the subunit 4 isoform 2 (COX IV-2) indicate involvement in the cellular response to hypoxia and oxidative stress. We evaluated whether the COX subunit IV isoform composition may explain the pronounced individual variations in resting metabolic rate (RMR). RMR was determined in healthy humans by indirect calorimetry and correlated to levels of COX IV-2 and COX IV-1 in vastus lateralis. Overexpression and knock down of the COX IV isoforms were performed in primary myotubes followed by evaluation of the cell respiration and production of reactive oxygen species. Here we show that COX IV-2 protein is constitutively expressed in human skeletal muscle and strongly correlated to RMR. Primary human myotubes overexpressing COX IV-2 displayed markedly (>60%) lower respiration, reduced (>50%) cellular H2O2 production, higher resistance toward both oxidative stress, and severe hypoxia compared with control cells. These results suggest an important role of isoform COX IV-2 in the control of energy expenditure, hypoxic tolerance, and mitochondrial ROS homeostasis in humans.

Renal denervation attenuates NADPH oxidase-mediated oxidative stress and hypertension in rats with hydronephrosis.

Hydronephrosis is associated with the development of salt-sensitive hypertension. Studies have suggested that increased sympathetic nerve activity and oxidative stress play important roles in hypertension and the modulation of salt sensitivity. The present study primarily aimed to examine the role of renal sympathetic nerve activity in the development of hypertension in rats with hydronephrosis. In addition, we aimed to investigate if NADPH oxidase (NOX) function could be affected by renal denervation. Partial unilateral ureteral obstruction (PUUO) was created in 3-wk-old rats to induce hydronephrosis. Sham surgery or renal denervation was performed at the same time. Blood pressure was measured during normal, high-, and low-salt diets. The renal excretion pattern, NOX activity, and expression as well as components of the renin-angiotensin-aldosterone system were characterized after treatment with the normal salt diet. On the normal salt diet, rats in the PUUO group had elevated blood pressure compared with control rats (115 ± 3 vs. 87 ± 1 mmHg, P < 0.05) and displayed increased urine production and lower urine osmolality. The blood pressure change in response to salt loading (salt sensitivity) was more pronounced in the PUUO group compared with the control group (15 ± 2 vs. 5 ± 1 mmHg, P < 0.05). Renal denervation in PUUO rats attenuated both hypertension (97 ± 3 mmHg) and salt sensitivity (5 ± 1 mmHg, P < 0.05) and normalized the renal excretion pattern, whereas the degree of renal fibrosis and inflammation was not changed. NOX activity and expression as well as renin and ANG II type 1A receptor expression were increased in the renal cortex from PUUO rats and normalized by denervation. Plasma Na(+) and K(+) levels were elevated in PUUO rats and normalized after renal denervation. Finally, denervation in PUUO rats was also associated with reduced NOX expression, superoxide production, and fibrosis in the heart. In conclusion, renal denervation attenuates hypertension and restores the renal excretion pattern, which is associated with reduced renal NOX and components of the renin-angiotensin-aldosterone system. This study emphasizes a link between renal nerves, the development of hypertension, and modulation of NOX function.

Abrogation of adenosine A1 receptor signalling improves metabolic regulation in mice by modulating oxidative stress and inflammatory responses.

AIMS/HYPOTHESIS: Adenosine is an important regulator of metabolism; however, the role of the A1 receptor during ageing and obesity is unclear. The aim of this study was to investigate the effects of A1 signalling in modulating metabolic function during ageing. METHODS: Age-matched young and aged A 1 (also known as Adora1)-knockout (A1(-/-)) and wild-type (A1(+/+)) mice were used. Metabolic regulation was evaluated by body composition, and glucose and insulin tolerance tests. Isolated islets and islet arterioles were used to detect islet endocrine and vascular function. Oxidative stress and inflammation status were measured in metabolic organs and systemically. RESULTS: Advanced age was associated with both reduced glucose clearance and insulin sensitivity, as well as increased visceral adipose tissue (VAT) in A1(+/+) compared with A1(-/-) mice. Islet morphology and insulin content were similar between genotypes, but relative changes in in vitro insulin release following glucose stimulation were reduced in aged A1(+/+) compared with A1(-/-) mice. Islet arteriolar responses to angiotensin II were stronger in aged A1(+/+) mice, this being associated with increased NADPH oxidase activity. Ageing resulted in multiple changes in A1(+/+) compared with A1(-/-) mice, including enhanced NADPH oxidase-derived O2(-) formation and NADPH oxidase isoform 2 (Nox2) protein expression in pancreas and VAT; elevated levels of circulating insulin, leptin and proinflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-12); and accumulation of CD4(+) T cells in VAT. This was associated with impaired insulin signalling in VAT from aged A1(+/+) mice. CONCLUSIONS/INTERPRETATION: These studies emphasise that A1 receptors regulate metabolism and islet endocrine and vascular functions during ageing, including via the modulation of oxidative stress and inflammatory responses, among other things.

Evaluation of two novel antioxidants with differential effects on curcumin-induced apoptosis in C2 skeletal myoblasts; involvement of JNKs.

Excessive levels of reactive oxygen species (ROS) result in numerous pathologies including muscle disorders. In essence, skeletal muscle performance of daily activities can be severely affected by the redox imbalances occurring after muscular injuries, surgery, atrophy due to immobilization, dystrophy or eccentric muscle contraction. Therefore, research on the potential beneficial impact of antioxidants is of outmost importance. In this context, aiming at further exploring the mechanisms of action of our newly synthesized antioxidant compounds (AK1 and AK2) in a skeletal muscle experimental setting, we initially investigated their scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and subsequently assessed their effect on the viability of C2 skeletal myoblasts in the presence of two pro-oxidants: H2O2 and curcumin (MTT assay). Interestingly, while both compounds reversed the detrimental effect of H2O2, only AK2 was cytoprotective in curcumin-treated C2 cells. We next confirmed the immediate activation of extracellular signal-regulated kinases (ERKs) and the more delayed activation profile of c-Jun NH2-terminal kinases (JNKs) in C2 skeletal myoblasts exposed to curcumin, by Western blotting. In correlation with the aforementioned results, only AK2 blocked the curcumin-induced activation of JNKs pathway. Furthermore, JNKs were revealed to mediate curcumin-induced apoptosis in C2 cells and only AK2 to effectively suppress it (by detecting its effect on poly(ADP-ribose) polymerase fragmentation). Overall, we have shown that two similar in structure novel antioxidants confer differential effects on C2 skeletal myoblasts viability under oxidative stress conditions. This result may be attributed to these antioxidants respective diverse mode of interaction with the signaling effectors involved in the observed responses. Future studies should further evaluate the mechanism of action of these compounds in order to support their potential application in therapeutic protocols against ROS-related muscle disorders.

Inhibition of cystathionine-gamma lyase dampens vasoconstriction in mouse and human intracerebral arterioles.

AIM: In extracerebral vascular beds cystathionine-gamma lyase (CSE) activity plays a vasodilatory role but the role of this hydrogen sulfide (H2 S) producing enzyme in the intracerebral arterioles remain poorly understood. We hypothesized a similar function in the intracerebral arterioles. METHODS: Intracerebral arterioles were isolated from wild type C57BL/6J mouse (9-12 months old) brains and from human brain biopsies. The function (contractility and secondary dilatation) of the intracerebral arterioles was tested ex vivo by pressure myography using a perfusion set-up. Reverse transcription polymerase chain reaction was used for detecting CSE expression. RESULTS: CSE is expressed in human and mouse intracerebral arterioles. CSE inhibition with L-propargylglycine (PAG) significantly dampened the K+ -induced vasoconstriction in intracerebral arterioles of both species (% of maximum contraction: in human control: 45.4 ± 2.7 versus PAG: 27 ± 5.2 and in mouse control: 50 ± 1.5 versus PAG: 33 ± 5.2) but did not affect the secondary dilatation. This effect of PAG was significantly reversed by the H2 S donor sodium hydrosulfide (NaSH) in human (PAG + NaSH: 38.8 ± 7.2) and mouse (PAG + NaSH: 41.7 ± 3.1) arterioles, respectively. The endothelial NO synthase (eNOS) inhibitor, Nω-Nitro-l-arginine methyl ester (L-NAME), and the inhibitor of soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of PAG on the K+ -induced vasoconstriction in the mouse arterioles and attenuated the K+ -induced secondary dilatation significantly. CONCLUSION: CSE contributes to the K+ -induced vasoconstriction via a mechanism involving H2 S, eNOS, and sGC whereas the secondary dilatation is regulated by eNOS and sGC but not by CSE.

Cystathionine gamma-lyase (CTH) inhibition attenuates glioblastoma formation.

PURPOSE: Glioblastoma (GBM) is the most common type of adult brain tumor with extremely poor survival. Cystathionine-gamma lyase (CTH) is one of the main Hydrogen Sulfide (H2S) producing enzymes and its expression contributes to tumorigenesis and angiogenesis but its role in glioblastoma development remains poorly understood. METHODS: and Principal Results: An established allogenic immunocompetent in vivo GBM model was used in C57BL/6J WT and CTH KO mice where the tumor volume and tumor microvessel density were blindly measured by stereological analysis. Tumor macrophage and stemness markers were measured by blinded immunohistochemistry. Mouse and human GBM cell lines were used for cell-based analyses. In human gliomas, the CTH expression was analyzed by bioinformatic analysis on different databases. In vivo, the genetic ablation of CTH in the host led to a significant reduction of the tumor volume and the protumorigenic and stemness transcription factor sex determining region Y-box 2 (SOX2). The tumor microvessel density (indicative of angiogenesis) and the expression levels of peritumoral macrophages showed no significant changes between the two genotypes. Bioinformatic analysis in human glioma tumors revealed that higher CTH expression is positively correlated to SOX2 expression and associated with worse overall survival in all grades of gliomas. Patients not responding to temozolomide have also higher CTH expression. In mouse or human GBM cells, pharmacological inhibition (PAG) or CTH knockdown (siRNA) attenuates GBM cell proliferation, migration and stem cell formation frequency. MAJOR CONCLUSIONS: Inhibition of CTH could be a new promising target against glioblastoma formation.

Hydrogen Sulfide and the Kidney: Physiological Roles, Contribution to Pathophysiology, and Therapeutic Potential.

Significance: Hydrogen sulfide (H2S), the third member of the gasotransmitter family, has a broad spectrum of biological activities, including antioxidant and cytoprotective actions, as well as vasodilatory, anti-inflammatory and antifibrotic effects. New, significant aspects of H2S biology in the kidney continue to emerge, underscoring the importance of this signaling molecule in kidney homeostasis, function, and disease. Recent Advances: H2S signals via three main mechanisms, by maintaining redox balance through its antioxidant actions, by post-translational modifications of cellular proteins (S-sulfhydration), and by binding to protein metal centers. Important renal functions such as glomerular filtration, renin release, or sodium reabsorption have been shown to be regulated by H2S, using either exogenous donors or by the endogenous-producing systems. Critical Issues: Lower H2S levels are observed in many renal pathologies, including renal ischemia-reperfusion injury and obstructive, diabetic, or hypertensive nephropathy. Unraveling the molecular targets through which H2S exerts its beneficial effects would be of great importance not only for understanding basic renal physiology, but also for identifying new pharmacological interventions for renal disease. Future Directions: Additional studies are needed to better understand the role of H2S in the kidney. Mapping the expression pattern of H2S-producing and -degrading enzymes in renal cells and generation of cell-specific knockout mice based on this information will be invaluable in the effort to unravel additional roles for H2S in kidney (patho)physiology. With this knowledge, novel targeted more effective therapeutic strategies for renal disease can be designed. Antioxid. Redox Signal. 36, 220-243.

Generation and Characterization of a CRISPR/Cas9 -Induced 3-mst Deficient Zebrafish.

3-mercaptopyruvate sulfurtransferase (3-MST) is an enzyme capable of synthesizing hydrogen sulfide (H2S) and polysulfides. In spite of its ubiquitous presence in mammalian cells, very few studies have investigated its contribution to homeostasis and disease development, thus the role of 3-MST remains largely unexplored. Here, we present a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) induced 3-mst mutant zebrafish line, which will allow the study of 3-MST's role in several biological processes. The 3-mst zebrafish orthologue was identified using a bioinformatic approach and verified by its ability to produce H2S in the presence of 3-mercaptopyruvate (3-MP). Its expression pattern was analyzed during zebrafish early development, indicating predominantly an expression in the heart and central nervous system. As expected, no detectable levels of 3-Mst protein were observed in homozygous mutant larvae. In line with this, H2S levels were reduced in 3-mst-/- zebrafish. Although the mutants showed no obvious morphological deficiencies, they exhibited increased lethality under oxidative stress conditions. The elevated levels of reactive oxygen species, detected following 3-mst deletion, are likely to drive this phenotype. In line with the increased ROS, we observed accelerated fin regenerative capacity in 3-mst deficient zebrafish. Overall, we provide evidence for the expression of 3-mst in zebrafish, confirm its important role in redox homeostasis and indicate the enzyme's possible involvement in the regeneration processes.

Cardiovascular phenotype of mice lacking 3-mercaptopyruvate sulfurtransferase.

RATIONALE: Hydrogen sulfide (H2S) is a physiological mediator that regulates cardiovascular homeostasis. Three major enzymes contribute to the generation of endogenously produced H2S, namely cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Although the biological roles of CSE and CBS have been extensively investigated in the cardiovascular system, very little is known about that of 3-MST. In the present study we determined the importance of 3-MST in the heart and blood vessels, using a genetic model with a global 3-MST deletion. RESULTS: 3-MST is the most abundant transcript in the mouse heart, compared to CSE and CBS. 3-MST was mainly localized in smooth muscle cells and cardiomyocytes, where it was present in both the mitochondria and the cytosol. Levels of serum and cardiac H2S species were not altered in adult young (2-3 months old) 3-MST-/- mice compared to WT animals. No significant changes in the expression of CSE and CBS were observed. Additionally, 3-MST-/- mice had normal left ventricular structure and function, blood pressure and vascular reactivity. Interestingly, genetic ablation of 3-MST protected mice against myocardial ischemia reperfusion injury, and abolished the protection offered by ischemic pre- and post-conditioning. 3-MST-/- mice showed lower expression levels of thiosulfate sulfurtransferase, lower levels of cellular antioxidants and elevated basal levels of cardiac reactive oxygen species. In parallel, 3-MST-/- mice showed no significant alterations in endothelial NO synthase or downstream targets. Finally, in a separate cohort of older 3-MST-/- mice (18 months old), a hypertensive phenotype associated with cardiac hypertrophy and NO insufficiency was observed. CONCLUSIONS: Overall, genetic ablation of 3-MST impacts on the mouse cardiovascular system in an age-dependent manner. Loss of 3-MST exerts a cardioprotective role in young adult mice, while with aging it predisposes them to hypertension and cardiac hypertrophy.

Genetic ablation of adenosine receptor A3 results in articular cartilage degeneration.

Osteoarthritis (OA), the most common form of arthritis, is characterized by inflammation of joints and cartilage degradation leading to disability, discomfort, severe pain, inflammation, and stiffness of the joint. It has been shown that adenosine, a purine nucleoside composed of adenine attached to ribofuranose, is enzymatically produced by the human synovium. However, the functional significance of adenosine signaling in homeostasis and pathology of synovial joints remains unclear. Adenosine acts through four cell surface receptors, i.e., A1, A2A, A2B, and A3, and here, we have systematically analyzed mice with a deficiency for A3 receptor as well as pharmacological modulations of this receptor with specific analogs. The data show that adenosine receptor signaling plays an essential role in downregulating catabolic mechanisms resulting in prevention of cartilage degeneration. Ablation of A3 resulted in development of OA in aged mice. Mechanistically, A3 signaling inhibited cellular catabolic processes in chondrocytes including downregulation of Ca2+/calmodulin-dependent protein kinase (CaMKII), an enzyme that promotes matrix degradation and inflammation, as well as Runt-related transcription factor 2 (RUNX2). Additionally, selective A3 agonists protected chondrocytes from cell apoptosis caused by pro-inflammatory cytokines or hypo-osmotic stress. These novel data illuminate the protective role of A3, which is mediated via inhibition of intracellular CaMKII kinase and RUNX2 transcription factor, the two major pro-catabolic regulators in articular cartilage. KEY MESSAGES: Adenosine receptor A3 (A3) knockout results in progressive loss of articular cartilage in vivo. Ablation of A3 results in activation of matrix degradation and cartilage hypertrophy. A3 agonists downregulate RUNX2 and CaMKII expression in osteoarthritic human articular chondrocytes. A3 prevents articular cartilage matrix degradation induced by inflammation and osmotic fluctuations. A3 agonist inhibits proteolytic activity of cartilage-degrading enzymes.

Adenosine signaling in diabetes mellitus and associated cardiovascular and renal complications.

Diabetes mellitus is characterized by abnormal glucose and lipid metabolism, and subsequent hyperglycemia and dyslipidemia, which results from defects in pancreatic islet beta-cells insulin secretion and/or decreased insulin sensitivity in metabolically active organs (i.e. liver, skeletal muscle and adipose tissue). Accumulating evidence highlights a critical role for the adenosine system in the regulation of insulin and glucose homeostasis and the pathophysiology of type 2 diabetes (T2D). Adenosine is a key diverse extracellular signaling molecule that regulates several aspects of tissue function by activating four G-protein-coupled receptors (i.e. A1, A2A, A2B and A3 receptors). Moreover, adenosine receptor signaling plays a critical role in inflammation, immune system, and oxidative stress, factors that are also important in metabolic disorders. This review discusses the role of the adenosine receptor system in the development or progression of diabetes mellitus, with specific focus on T2D, and associated complications linked to the cardiovascular and renal systems.

Pharmacological targeting of adenosine receptor signaling.

Adenosine receptor signaling plays important roles in normal physiology, but is also known to modulate the development or progression of several different diseases. The design of new, efficient, and safe pharmacological approaches to target the adenosine system may have considerable therapeutic potential, but is also associated with many challenges. This review summarizes the main challenges of adenosine receptor targeted treatment including tolerance, disease stage, cell type-specific effects, caffeine intake, adenosine level assessment and receptor distribution in vivo. Moreover, we discuss several potential ways to overcome these obstacles (i.e., the use of partial agonists, indirect receptor targeting, allosteric enhancers, prodrugs, non-receptor-mediated effects, neoreceptors, conditional knockouts). It is important to address these concerns during development of new and successful therapeutic approaches targeting the adenosine system.

Renal denervation attenuates hypertension and renal dysfunction in a model of cardiovascular and renal disease, which is associated with reduced NADPH and xanthine oxidase activity.

Oxidative stress is considered a central pathophysiological event in cardiovascular disease, including hypertension. Early age reduction in renal mass is associated with hypertension and oxidative stress in later life, which is aggravated by increased salt intake. The aim of the present study was to examine if renal sympathetic denervation can exert blood pressure lowering effects in uninephrectomized (UNX) rats (3-week old) fed with high salt (HS, 4%; w/w) diet for 4 weeks. Moreover, we investigated if renal denervation is associated with changes in NADPH and xanthine oxidase-derived reactive oxygen species. Rats with UNX + HS had reduced renal function, elevated systolic and diastolic arterial pressures, which was accompanied by increased heart weight, and cardiac superoxide production compared to sham operated Controls. UNX + HS was also associated with higher expression and activity of NADPH and xanthine oxidase in the kidney. Renal denervation in rats with UNX + HS attenuated the development of hypertension and cardiac hypertrophy, but also improved glomerular filtration rate and reduced proteinuria. Mechanistically, renal denervation was associated with lower expression and activity of both NADPH oxidase and xanthine oxidase in the kidney, but also reduced superoxide production in the heart. In conclusion, our study shows for the first time that renal denervation has anti-hypertensive, cardio- and reno-protective effects in the UNX + HS model, which can be associated with decreased NADPH oxidase- and xanthine oxidase-derived reactive oxygen species (i.e., superoxide and hydrogen peroxide) in the kidney.

Dietary nitrate attenuates renal ischemia-reperfusion injuries by modulation of immune responses and reduction of oxidative stress.

Ischemia-reperfusion (IR) injury involves complex pathological processes in which reduction of nitric oxide (NO) bioavailability is suggested as a key factor. Inorganic nitrate can form NO in vivo via NO synthase-independent pathways and may thus provide beneficial effects during IR. Herein we evaluated the effects of dietary nitrate supplementation in a renal IR model. Male mice (C57BL/6J) were fed nitrate-supplemented chow (1.0mmol/kg/day) or standard chow for two weeks prior to 30min ischemia and during the reperfusion period. Unilateral renal IR caused profound tubular and glomerular damage in the ischemic kidney. Renal function, assessed by plasma creatinine levels, glomerular filtration rate and renal plasma flow, was also impaired after IR. All these pathologies were significantly improved by nitrate. Mechanistically, nitrate treatment reduced renal superoxide generation, pro-inflammatory cytokines (IL-1ß, IL-6 and IL-12 p70) and macrophage infiltration in the kidney. Moreover, nitrate reduced mRNA expression of pro-inflammatory cytokines and chemo attractors, while increasing anti-inflammatory cytokines in the injured kidney. In another cohort of mice, two weeks of nitrate supplementation lowered superoxide generation and IL-6 expression in bone marrow-derived macrophages. Our study demonstrates protective effect of dietary nitrate in renal IR injury that may be mediated via modulation of oxidative stress and inflammatory responses. These novel findings suggest that nitrate supplementation deserve further exploration as a potential treatment in patients at high risk of renal IR injury.

Dietary nitrate improves age-related hypertension and metabolic abnormalities in rats via modulation of angiotensin II receptor signaling and inhibition of superoxide generation.

Advanced age is associated with increased risk for cardiovascular disease and type 2 diabetes. A proposed central event is diminished amounts of nitric oxide (NO) due to reduced generation by endothelial NO synthase (eNOS) and increased oxidative stress. In addition, it is widely accepted that increased angiotensin II (ANG II) signaling is also implicated in the pathogenesis of endothelial dysfunction and hypertension by accelerating formation of reactive oxygen species. This study was designed to test the hypothesis that dietary nitrate supplementation could reduce blood pressure and improve glucose tolerance in aged rats, via attenuation of NADPH oxidase activity and ANG II receptor signaling. Dietary nitrate supplementation for two weeks reduced blood pressure (10-15mmHg) and improved glucose clearance in old, but not in young rats. These favorable effects were associated with increased insulin responses, reduced plasma creatinine as well as improved endothelial relaxation to acetylcholine and attenuated contractility to ANG II in resistance arteries. Mechanistically, nitrate reduced NADPH oxidase-mediated oxidative stress in the cardiovascular system and increased cGMP signaling. Finally, nitrate treatment in aged rats normalized the gene expression profile of ANG II receptors (AT1A, AT2, AT1A/AT2 ratio) in the renal and cardiovascular systems without altering plasma levels of renin or ANG II. Our results show that boosting the nitrate-nitrite-NO pathway can partly compensate for age-related disturbances in endogenous NO generation via inhibition of NADPH oxidase and modulation of ANG II receptor expression. These novel findings may have implications for nutrition-based preventive and therapeutic strategies against cardiovascular and metabolic diseases.

Enhanced XOR activity in eNOS-deficient mice: Effects on the nitrate-nitrite-NO pathway and ROS homeostasis.

Xanthine oxidoreductase (XOR) is generally known as the final enzyme in purine metabolism and as a source of reactive oxygen species (ROS). In addition, this enzyme has been suggested to mediate nitric oxide (NO) formation via reduction of inorganic nitrate and nitrite. This NO synthase (NOS)-independent pathway for NO generation is of particular importance during certain conditions when NO bioavailability is diminished due to reduced activity of endothelial NOS (eNOS) or increased oxidative stress, including aging and cardiovascular disease. The exact interplay between NOS- and XOR-derived NO generation is not fully elucidated yet. The aim of the present study was to investigate if eNOS deficiency is associated with changes in XOR expression and activity and the possible impact on nitrite, NO and ROS homeostasis. Plasma levels of nitrate and nitrite were similar between eNOS deficient (eNOS-/-) and wildtype (wt) mice. XOR activity was upregulated in eNOS-/- compared with wt, but not in nNOS-/-, iNOS-/- or wt mice treated with the non-selective NOS inhibitor L-NAME. Following an acute dose of nitrate, plasma nitrite increased more in eNOS-/- compared with wt, and this augmented response was abolished by the selective XOR inhibitor febuxostat. Livers from eNOS-/- displayed higher nitrite reducing capacity compared with wt, and this effect was attenuated by febuxostat. Dietary supplementation with nitrate increased XOR expression and activity, but concomitantly reduced superoxide generation. The latter effect was also seen in vitro after nitrite administration. Treatment with febuxostat elevated blood pressure in eNOS-/-, but not in wt mice. A high dose of dietary nitrate reduced blood pressure in naïve eNOS-/- mice, and again this effect was abolished by febuxostat. In conclusion, eNOS deficiency is associated with an upregulation of XOR facilitating the nitrate-nitrite-NO pathway and decreasing the generation of ROS. This interplay between XOR and eNOS is proposed to play a significant role in NO homeostasis and blood pressure regulation.

Nitric oxide generation by the organic nitrate NDBP attenuates oxidative stress and angiotensin II-mediated hypertension.

BACKGROUND AND PURPOSE: NO deficiency and oxidative stress are crucially involved in the development or progression of cardiovascular disease, including hypertension and stroke. We have previously demonstrated that acute treatment with the newly discovered organic nitrate, 2-nitrate-1,3-dibuthoxypropan (NDBP), is associated with NO-like effects in the vasculature. This study aimed to further characterize the mechanism(s) and to elucidate the therapeutic potential in a model of hypertension and oxidative stress. EXPERIMENTAL APPROACH: A combination of ex vivo, in vitro and in vivo approaches was used to assess the effects of NDBP on vascular reactivity, NO release, NADPH oxidase activity and in a model of hypertension. KEY RESULTS: Ex vivo vascular studies demonstrated NDBP-mediated vasorelaxation in mesenteric resistance arteries, which was devoid of tolerance. In vitro studies using liver and kidney homogenates revealed dose-dependent and sustained NO generation by NDBP, which was attenuated by the xanthine oxidase inhibitor febuxostat. In addition, NDBP reduced NADPH oxidase activity in the liver and prevented angiotensin II-induced activation of NADPH oxidase in the kidney. In vivo studies showed that NDBP halted the progression of hypertension in mice with chronic angiotensin II infusion. This was associated with attenuated cardiac hypertrophy, and reduced NADPH oxidase-derived oxidative stress and fibrosis in the kidney and heart. CONCLUSION AND IMPLICATIONS: The novel organic nitrate NDBP halts the progression of angiotensin II-mediated hypertension. Mechanistically, our findings suggest that NDBP treatment is associated with sustained NO release and attenuated activity of NADPH oxidase, which to some extent requires functional xanthine oxidase.

Nitrite-mediated renal vasodilatation is increased during ischemic conditions via cGMP-independent signaling.

The kidney is vulnerable to hypoxia, and substantial efforts have been made to ameliorate renal ischemic injury secondary to pathological conditions. Stimulation of the nitrate-nitrite-nitric oxide pathway is associated with renal and cardiovascular protection in disease models, but less is known about the vascular effects during renal ischemia. This study was aimed at investigating the vascular effects of nitrite in the kidney during normoxic and ischemic conditions. Using a multiwire myograph system, we assessed nitrite-mediated relaxation (10(-9)-10(-4)mol/L) in isolated and preconstricted renal interlobar arteries from C57BL/6 mice under normal conditions (pO2 13kPa; pH 7.4) and with low oxygen tension and low pH to mimic ischemia (pO2 3kPa; pH 6.6). Xanthine oxidoreductase expression was analyzed by quantitative PCR, and production of reactive nitrogen species was measured by DAF-FM DA fluorescence. During normoxia significant vasodilatation (15±3%) was observed only at the highest concentration of nitrite, which was dependent on NO-sGC-cGMP signaling. The vasodilatory responses to nitrite were greatly sensitized and enhanced during hypoxia with low pH, demonstrating significant dilatation (11±1%) already in the physiological range (10(-8)mol/L), with a maximum response of 27±2% at 10(-4) mol/L. In contrast to normoxia, and to that observed with a classical NO donor (DEA NONOate), this sensitization was independent of sGC-cGMP signaling. Moreover, inhibition of various enzymatic systems reported to reduce nitrite in other vascular beds, i.e., aldehyde oxidase (raloxifene), aldehyde dehydrogenase (cyanamide), and NO synthase (L-NAME), had no effect on the nitrite response. However, inhibition of xanthine oxidoreductase (XOR; febuxostat or allopurinol) abolished the sensitized response to nitrite during hypoxia and acidosis. In conclusion, in contrast to normoxia, nitrite exerted potent vasorelaxation during ischemic conditions already at physiological concentrations. This effect was dependent on functional XOR but independent of classical downstream signaling by sGC-cGMP.