NADPH oxidase and xanthine oxidoreductase as targets and regulators of the nitrate-nitrite-nitric oxide pathway

Overproduction of reactive oxygen species (ROS) by NADPH oxidase (NOX) and xanthine oxidoreductase (XOR) with a concomitant decrease in the bioavailability of nitric oxide (NO) from eNOS contributes to the development of cardiovascular and metabolic disorders. Extensive research has proven the existence of an alternative NOS-independent pathway for NO production. This nitrate-nitrite-NO pathway starts with inorganic nitrate which is derived from the diet and from oxidized NO. Nitrate is reduced to nitrite mainly by oral commensal bacteria and then to NO and other bioactive nitrogen oxides in blood and tissues. The aim of the current thesis was to investigate the therapeutic role of the nitrate-nitrite-NO pathway in models of hypertension, metabolic dysfunction and inflammation focusing on whether the main ROS-producing enzymes NOX and XOR could be targets or even regulators of the this pathway. We show that NOX in the renal microvasculature is a primary target for the blood pressure lowering effects of dietary nitrate in Angiotensin-II (AngII)-mediated hypertension. In agreement, NOX activity and AngII-induced receptor signaling are downregulated by nitrate in aged and hypertensive rats. Moreover, nitrate targets the elevated liver NOX activity of aged and metabolically dysregulated mice with an improvement of AMPK activity. Finally, we show that nitrite can act on cells of the innate immune response. In particular, NOX mediated superoxide production is strongly attenuated in activated macrophages with a concomitant reduction on iNOS gene expression and peroxynitrite production. Mechanistically, we observed that symbiotic bacteria and XOR together are responsible for the bioactivation of dietary nitrate to form NO. In addition, the effects of nitrate and nitrite are not only NOS independent but actually potentiated when eNOS activity is pharmacologically, genetically or naturally impaired. Interestingly, the absence of eNOS is associated with a higher XOR activity that partly compensates for the disrupted NO homeostasis and elevated blood pressure in these mice. Also, the blood pressure response to dietary nitrate is augmented in eNOS-/- mice and abolished upon XOR inhibition. Interestingly, nitrate and nitrite were able to switch the function of XOR towards lower ROS and higher NO production which could significantly contribute to the antihypertensive effects. In conclusion, administration of nitrate or nitrite is associated with a number of therapeutic cardiovascular and metabolic effects in animal models of disease. We propose that NOX and XOR are two main targets and possible regulators of the nitrate-nitrite-NO pathway. This pathway is triggered in situations with higher NOX and XOR activity and acts in parallel with the NOS dependent NO production to uphold NO homeostasis

Monitoring temporal variations in the geothermal activity of Miocene Lesvos volcanic field using remote sensing techniques and MODIS - LST imagery

Abstract Many islands of the Aegean Sea show strong geothermal activity due to volcanism in the area. In this paper, Robust Satellite Techniques (RST) are used to isolate, from other known possible sources, those thermal anomalies possibly related to geothermal activity in the Miocene volcanic field of Lesvos Island (Northern Aegean). For this purpose, 12 years (2003–2014) of daily Night-time Land Surface Temperature (LST) products, from Moderate Resolution Imaging Spectroradiometer (MODIS) acquisitions were analyzed. The final dataset contained 770 thermal anomalies whose spatial correlation with geological and structural data of Lesvos - such as caldera rims, ring faults, major tectonic lineaments and hydrothermal alterations mapped by processing SENTINEL-2 MSI satellite images – has been particularly investigated. In the approximately 20 ma geothermal field of Lesvos, geothermal activity seems to be also associated with the extensional regime of the broader area that leads to lithosphere thinning and consequent heat transfer in the multi-fractured terrain of Lesvos through volcanic and tectonic faults. Achieved results seem to confirm the possibility to use RST-based thermal anomalies to identify temporal variations in the geothermal activity probably due to the uplifting and circulation of the hydrothermal waters

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

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 β-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

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.</p

Satellite-Observed Thermal Anomalies and Deformation Patterns Associated to the 2021, Central Crete Seismic Sequence

Nowadays, there has been a growing interest in understanding earthquake forerunners, i.e., anomalous variations that are possibly associated with the complex process of earthquake evolution. In this context, the Robust Satellite Technique was coupled with 10 years (2012–2021) of daily night-time MODIS-Land Surface Temperature remote sensing data to detect thermal anomalies likely related to the 27 September 2021, strong onshore earthquake of magnitude Mw6.0 occurring near the Arkalochori village in Central Crete, Greece. Eight intense (signal-to-noise ratio > 3) and infrequent, quite extensive, and temporally persistent thermal signal transients were detected and characterized as pre-seismic anomalies, while one thermal signal transient was identified as a co-seismic effect on the day of the main tectonic event. The thermal anomalies dataset was combined with tectonic parameters of Central Crete, such as active faults and fault density, seismogenic zones and ground displacement maps produced using Sentinel-1 satellite imagery and the Interferometric Synthetic Aperture Radar technique. Regarding the thermal anomaly of 27 September, its greatest portion was observed over the footwall part of the fault where a significant subsidence up to 20 cm exists. We suggest that the thermal anomalies are possibly connected with gas release which happens due to stress changes and is controlled by the existence of tectonic lines and the density of the faults, even if alternative explanations could not be excluded

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

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-beta), (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

In adenosine A(2B) knockouts acute treatment with inorganic nitrate improves glucose disposal, oxidative stress, and AMPK signaling in the liver

Rationale: Accumulating studies suggest that nitric oxide (NO) deficiency and oxidative stress are central pathological mechanisms in type 2 diabetes (T2D). Recent findings demonstrate therapeutic effects by boosting the nitrate-nitrite-NO pathway, which is an alternative pathway for NO formation. This study aimed at investigating the acute effects of inorganic nitrate on glucose and insulin signaling in adenosine A2B receptor knockout mice (A(2B)(-/-), a genetic mouse model of impaired metabolic regulation. Methods: Acute effects of nitrate treatment were investigated in aged wild-type (WT) and A(2B)(-/-) mice. One hour after injection with nitrate (0.1 mmol/kg, i.p.) or placebo, metabolic regulation was evaluated by intraperitoneal glucose and insulin tolerance tests. NADPH oxidase-mediated superoxide production and AMPK phosphorylation were measured in livers obtained from non-treated or glucose-treated mice, with or without prior nitrate injection. Plasma was used to determine insulin resistance (HOMA-IR) and NO signaling. Results: A(2B)(-/-) displayed increased body weight, reduced glucose clearance, and attenuated overall insulin responses compared with age-matched WT mice. Nitrate treatment increased circulating levels of nitrate, nitrite and cGMP in the A(2B)(-/-), and improved glucose clearance. In WT mice, however, nitrate treatment did not influence glucose clearance. HOMA-IR increased following glucose injection in the A(2B)(-/-), but remained at basal levels in mice pretreated with nitrate. NADPH oxidase activity in livers from A(2B)(-/-), but not WT mice, was reduced by nitrate treatment. Livers from A(2B)(-/-) displayed reduced AMPK phosphorylation compared with WT mice, and this was increased by nitrate treatment. Finally, injection with the anti-diabetic agent metformin induced similar therapeutic effects in the A(2B)(-/-) as observed with nitrate. Conclusion: The A(2B)(-/-) mouse is a genetic mouse model of metabolic syndrome. Acute treatment with nitrate improved the metabolic profile in it, at least partly via reduction in oxidative stress and improved AMPK signaling in the liver