Protective effect of trans-nerolidol on vascular endothelial cell injury induced by lipopolysaccharides / Efectos protectores del trans-nerolidol en lesiones inducidas por lipopolisacáridos en células endoteliales vasculares

This article aimed to discuss the protection of trans - nerolidol on vascular endothelial cells (ECs) injured by lipopolysac charides. ECs were divided into four groups: normal, model, low and high dose trans - nerolidol treatment groups. The cell survival rate and the contents of NO in the cell culture supernatant were determined. The protein expression and transcript level of pe roxisome proliferator - activated receptor - γ (PPARγ), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were determined by western blotting and RT - PCR respectively. Compared with the normal group, cell livability, protein e xpression and mRNA transcript level of PPARγ and eNOS decreased, NO contents, protein expression and mRNA transcript tlevel of iNOS increased in model group significantly. Compared with model group, all the changes recovered in different degree in treatmen t groups. Hence, it was concluded that trans - nerolidol can alleviate the ECs injuryby the regulation of iNOS/eNOS through activating PPARγ in a dose - dependent manner

Este artículo tiene como objetivo discutir la protección del trans - nerolidol en las células endoteliales vasculares (CE) dañadas por lipopolisacáridos. Las CE se di vidieron en cuatro grupos: normal, modelo, grupos de tratamiento con trans - nerolidol de baja y alta dosis. Se determinó la tasa de supervivencia de las células y los contenidos de óxido nítrico (NO) en el sobrenadante del cultivo celular. La expresión de p roteínas y el nivel de transcripción del receptor activado por proliferadores de peroxisomas - γ (PPARγ), el óxido nítrico sint et asa endotelial (eNOS) y el óxido nítrico sint et asa inducible (iNOS) se determinaron mediante western blot y RT - PCR, respectivamen te. En comparación con el grupo normal, la viabilidad celular, la expresión de proteínas y el nivel de transcripción de PPARγ y eNOS disminuyeron, los contenidos de NO, la expresión de proteínas y el nivel de transcripción de iNOS aumentaron significativam ente en el grupo modelo. En comparación con el grupo modelo, todos los cambios se recuperaron en diferentes grados en los grupos de tratamiento. Por lo tanto, se concluyó que el trans - nerolidol puede aliviar el daño en las CE regulando iNOS/eNOS a través d e la activación de PPARγ de manera dependiente de la dosis.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide.

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Hyaluronic Acid-Coated Silica Nanoparticles for Targeted Delivery of Nitric Oxide to Cancer Cells.

Drug resistance and off-target toxicity are two of the greatest challenges to chemotherapeutic melanoma treatments. Nitric oxide (NO) represents an attractive alternative to conventional therapeutics due to its numerous anticancer properties and low probability of engendering resistance. As NO is highly reactive, macromolecular NO donors are needed for the controlled and targeted delivery of NO for therapeutic applications. Herein, mesoporous silica nanoparticles (MSNs) coated with hyaluronic acid (HA) were developed as a NO delivery system to facilitate controlled delivery to cancer cells through both passive and active targeting via the enhanced permeation and retention effect and directed binding of HA with CD44 receptors, respectively. The aminosilane modification, HA concentration, and HA molecular weight were systematically evaluated to facilitate the MSN coating and NO loading. The hydrodynamic diameter and dispersity of the nanoparticles increased after HA coating due to the hydrophilic nature of HA, with greater increases observed at higher HA molecular weight. Lower starting concentrations of HA and aminosilanes with longer alkyl chains favored more efficient HA coating. Faster NO-release kinetics and lower NO payloads were observed for the HA-coated MSNs relative to uncoated MSNs. However, the localized delivery of NO to cancer cells through the active targeting conferred by HA increased levels of oxidative stress and induced mitochondria-mediated apoptosis in melanoma cells. Cytotoxicity was also evaluated against human dermal fibroblasts, with the use of 6 kDa HA-coated MSNs resulting in the greatest therapeutic indices. Enhanced internalization of HA-coated nanoparticles into melanoma cells versus uncoated nanoparticles was visualized with confocal microscopy and quantified by fluorescence spectroscopy. In total, HA-coated MSNs represent a promising NO delivery system for potential use as a chemotherapeutic for skin melanomas.

Pro-inflammatory properties of aureocin A53.

Aureocin A53 is a peptide bacteriocin produced by an opportunistic pathogen Staphylococcus aureus strain A53. The spatial structure of aureocin, unlike its amino acid sequence, is similar to the bacteriocin BacSp222, which was recently found to have the ability to induce the inflammatory response in the host cells. The presented research aimed to verify such properties also for aureocin A53. We demonstrated that the synthetic aureocin has slight cytotoxic activity towards murine monocytic-macrophage cells. This molecule was also able to activate murine P388.D1 and RAW 264.7 cells to IFN-γ-dependent production of nitric oxide and to activate production of the pro-inflammatory cytokine - TNF. We also proved that the observed pro-inflammatory activity of the studied bacteriocin is related to the stimulation of the TLR2/TLR6 heterodimer and, consequently, activation of the NF-κB transcription factor. To sum up, A53 is the second bacteriocin described in the literature, showing the pro-inflammatory activity against murine macrophage-like cells.

Fractional exhaled nitric oxide distribution and its relevant factors in the general adult population and its healthy subpopulation.

Background: Measurement of fractional exhaled nitric oxide (Feno) has been used in the diagnosis and management of asthma. Understanding the distribution of Feno in a larger resident population and its "healthy" subpopulation would contribute to the interpretation of Feno in clinical practice. Objective: This study aimed to investigate the distribution and its associated factors in the adult population and its healthy subpopulations. Methods: We conducted a cross-sectional study of 8,638 men and 17,288 women aged 20 years or older living in Miyagi prefecture, Japan. We investigated the distribution of Feno and its associated factors in all subjects, a subpopulation with no history of upper and lower airway diseases (healthy subpopulation 1), and a subpopulation with no history of upper and lower airway diseases, normal lung function, and no positivity for other biomarkers of type 2 inflammation (healthy subpopulation 2). Results: The distribution of Feno in healthy subpopulations, especially in healthy subpopulation 2 (median [interquartile range], 17 [12-23] with 95th percentile of 36 ppb) was lower than in all subjects (19 [13-26] ppb with 95th percentile of 47 ppb). In healthy subpopulation 1, 10.3% had elevated Feno (≥35 ppb), and elevated Feno was positively associated with factors including obstructive ventilatory defect, blood eosinophilia, house dust mite-specific IgE positivity, and history of hypertension. Male sex was associated with elevated Feno in all subjects and healthy subpopulations. Conclusion: The distribution of Feno in the healthy subpopulation supports the validity of the criteria (≥35 ppb) currently used in Japan for the diagnosis of asthma.

The effect of spexin injection and its interaction with nitric oxide, serotonin, and corticotropin receptors on the central regulation of food intake in broilers.

Complex homeostatic control mechanisms are tools to adjust the food birds eat and their appetite. Birds and mammals differ in several ways considering food intake regulation. Therefore, this study aimed to investigate the special effects of the intracerebroventricular (ICV) injection of spexin and its interaction with nitric oxide, serotonin and corticotropin receptors on central food intake regulation in broilers. In the test 1, Broilers received ICV injection of saline, PCPA (p-chlorophenylalanine,1.25 µg), spexin (10 nmol) and PCPA+spexin. In test 2-7, 8-OH-DPAT, SB-242084 (5-HT2C, 1.5 µg), L-arginine (Precursor of nitric oxide, 200 nmol), L-NAME (nitric oxide synthetize inhibitor, 100 nmol), Astressin-B (30 µg) and Astressin2-B (30 µg) were injected to Broilers instead of the PCPA. Then, the amount of food received was measured up to 2 h after the injection. The food consumption was significantly decreased by Spexin (10 nmol) (P<0.05). Concomitant injection of SB-242084+spexin attenuated spexin-induced hypophagia (P<0.05). Co-injection of L-arginine+spexin enhanced spexin-induced hypophagia and this effect was reversed by L-NAME (P<0.05). Also, concomitant injection of Astressin-B + spexin or Astressin2-B + spexin enhanced spexin-induced hypophagia (P<0.05). Founded on these observations, spexin-induced hypophagia may be mediated by nitric oxide and 5-HT2C, CRF1, and CRF2 receptors in neonatal broilers.

Towards rational design in electrochemical denitrification by analyzing pH dependence.

A small fraction of NOx (<1%) always exists in CO2 feedstock (e.g. exhausted gas), which can significantly reduce the efficiency of CO2 electroreduction by ∼30%. Hence, electrochemical denitrification is the precondition of CO2 electroreduction. The pH effect is a key factor, and can be used to tune the selectivity between N2 and N2O production in electrochemical denitrification. However, there has been much controversy for many years about the origin of pH dependence in electrocatalysis. To this end, we present a new scheme to accurately model the pH dependence of the electrochemical mechanism. An extremely small pH variation from pH 12.7 to pH 14 can be accurately reproduced for N2O production. More importantly, the obviously different pH dependence of N2 production, compared to N2O, can be attributed to a cascade path. In other words, the N2 was produced from the secondary conversion of the as-produced N2O molecule (the major product), instead of the original reactant NO. This is further supported by more than 35 experiments over varying catalysts (Fe, Ni, Pd, Cu, Co, Pt and Ag), partial pressures (20%, 50% and 100%) and potentials (from -0.2 to 0.2 V vs. reversible hydrogen electrode). All in all, the insights herein overturn long-lasting views in the field of NO electroreduction and suggest that rational design should steer away from catalyst engineering toward reactor optimization.

S-nitrosylation may inhibit the activity of COP1 in plant photomorphogenesis.

Protein S-nitrosylation, which is defined by the covalent attachment of nitric oxide (NO) to the thiol group of cysteine residues, is known to play critical roles in plant development and stress responses. NO promotes seedling photomorphogenesis and NO emission is enhanced by light. However, the function of protein S-nitrosylation in plant photomorphogenesis is largely unknown. E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) antagonistically regulate seedling photomorphogenesis. COP1 inhibits plant photomorphogenesis by targeting photomorphogenic promoters like HY5 for 26S proteasome degradation. Here, we report that COP1 is S-nitrosylated in vitro. Mass spectrometry analyses revealed that two evolutionarily well conserved residues, cysteine 425 and cysteine 607, in the WD40 domain of COP1 are S-nitrosylated. S-nitrosylated glutathione (GSNO) is an important physiological NO donor for protein S-nitrosylation. The Arabidopsis (Arabidopsis thaliana) gsnor1-3 mutant, which accumulates higher level of GSNO, accumulated higher HY5 levels than wildtype (WT), indicating that COP1 activity is inhibited. Protein S-nitrosylation can be reversed by Thioredoxin-h5 (TRXh5) in plants. Indeed, COP1 interacts directly with TRXh5 and its close homolog TRXh3. Moreover, catalase 3 (CAT3) acts as a transnitrosylase that transfers NO to its target proteins like GSNO reductase (GSNOR). We found that CAT3 interacts with COP1 in plants. Taken together, our data indicate that the activity of COP1 is likely inhibited by NO via S-nitrosylation to promote the accumulation of HY5 and photomorphogenesis.

Spermidine Exerts Protective Effects in Random-Pattern Skin Flap Survival in Rats: Possible Involvement of Inflammatory Cytokines, Nitric Oxide, and VEGF.

BACKGROUND: Distal necrosis and inflammation are two of the most common health consequences of random-pattern skin flaps survival (SFS). Anti-inflammatory effects of spermidine have been identified in various studies. On the other hand, considering the involvement of the nitric oxide molecule in the spermidine mode of action and also its role in skin tissue function, we analyzed the possible effects of spermidine on the SFS and also, potential involvement of nitrergic pathway and inflammatory cytokine in these phenomena. METHODS: Each rat was pretreated with either a vehicle (control) or various doses of spermidine (0.5, 1, 3, 5, 10 and 30 mg/kg) and then was executed a random-pattern skin flap paradigm. Also, spermidine at the dose of 5 mg/kg was selected and one group rats received spermidine 20 min prior to surgery and one additional dose 1 day after operation. Then, 7 days after operations, interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon-gamma (IFN-γ), and nitrite levels were inquired in the tissue samples by ELIZA kit. Vascular endothelial growth factor expression was assessed by DAPI staining and fluorescent microscopes. The concentrations of three polyamines, including spermidine, spermine, and cadaverine, were analyzed using HPLC. RESULTS: Pretreatment with spermidine 5 mg/kg improved SFS considerably in microscopic skin H&E staining analysis and decreased the percentage of necrotic area. Moreover, spermidine exerted promising anti-inflammatory effects via the modulation of nitric oxide and reducing inflammatory cytokines. CONCLUSIONS: Spermidine could improve skin flaps survival, probably through the nitrergic system and inflammation pathways. This preclinical study provides level III evidence for the potential therapeutic effects of spermidine on SFS in rats, based on the analysis of animal models. Further studies are needed to confirm these findings in clinical settings. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Isoflurane increases the activity of the vascular matrix metalloproteinase-2 in non-pregnant rats and increases the nitric oxide metabolites in pregnancy.

Surgeries that require general anesthesia occur in 1.5-2% of gestations. Isoflurane is frequently used because of its lower possibility of affecting fetal growth. Therefore, we examined the isoflurane anesthesia-induced effects on maternal hemodynamic and vascular changes. We hypothesized that isoflurane would enhance endothelium-dependent vasodilation as a consequence of increased nitric oxide and decreased metalloproteinases (MMPs). Female rats (n = 28) were randomized into 4 groups (7 rats/group): conscious (non-anesthetized) non-pregnant group, non-pregnant anesthetized group, conscious pregnant group, and pregnant anesthetized group. Anesthesia was performed on the 20th pregnancy day, and hemodynamic parameters were monitored. Nitric oxide metabolites, gelatinolytic activity of MMP-2 and MMP-9, and the vascular function were assessed. Isoflurane caused no significant hemodynamic changes in pregnant compared with non-pregnant anesthetized group. Impaired acetylcholine-induced relaxations were observed only in conscious non-pregnant group (by approximately 62%) vs 81% for other groups. Phenylephrine-induced contractions were greater in endothelium-removed aorta segments of both pregnant groups (with or without isoflurane) compared with non-pregnant groups. Higher nitric oxide metabolites were observed in anesthetized pregnant in comparison with the other groups. Reductions in the 75 kDa activity and concomitant increases in 64 kDa MMP-2 isoforms were observed in aortas of pregnant anesthetized (or not) groups compared with conscious non-pregnant group. Isoflurane anesthesia shows stable effects on hemodynamic parameters and normal MMP-2 activation in pregnancy. Furthermore, there were increases in nitric oxide bioavailability, suggesting that isoflurane provides protective actions to the endothelium in pregnancy.

Blood Level of Nitric Oxide Metabolites and Expression of Genes Regulating NO Synthesis in Early Forms of Non-Alcoholic Fatty Liver Disease.

The levels of NO metabolites in the plasma and mRNA of the NOS3, ATG9B, and NOS2 genes in peripheral blood leukocytes of healthy people and patients with early forms of non-alcoholic fatty liver disease (steatosis and weak activity non-alcoholic steatohepatitis) were studied. In patients with steatohepatitis, the concentration of NO metabolites in the blood and the level of mRNA of the NOS2 gene were higher than in patients with steatosis and healthy people. These differences can be of diagnostic value for distinguishing between steatosis and weak activity steatohepatitis in non-alcoholic fatty liver disease. A correlation between the levels of NO metabolites and the expression of the NOS2 gene in weak activity steatohepatitis was established, which indicates activation of NO synthesis in non-alcoholic steatohepatitis due to the expression of the inducible NO synthase gene. The level of the NOS2 gene mRNA in peripheral blood leukocytes of patients with weak activity steatohepatitis correlated with the level of TNFα and IL-6 cytokines. An increase in the level of NO in the blood in weak activity steatohepatitis correlated with the level of MDA, an indicator of oxidative stress.

Hypoxia-induced NOS1 as a therapeutic target in hypercholesterolemia-related colorectal cancer.

BACKGROUND: It is well established that hypercholesterolemia increases the risk of atherosclerosis, especially because it reduces the availability of nitric oxide (NO). However, the relationship between hypercholesterolemia and NO in regulating colorectal cancer development and progression remains unknown. METHODS: We conducted bioinformatics analysis, qRT-PCR, ChIP-qPCR assays, luciferase report assays, clonogenic survival assays, and multiple mouse models to investigate the function and mechanism of hypercholesterolemia in regulating NO signaling. Additionally, NOS inhibitors were used to evaluate the potential of therapeutic strategy in anti-tumor response. RESULTS: Here, we show that oxidized low-density lipoprotein (oxLDL) cholesterol and its receptor LOX-1 are essential for hypercholesterolemia-induced colorectal tumorigenesis. Mechanically, the oxLDL promotes the oxidant stress-dependent induction of hypoxia signaling to transcriptionally up-regulate NO synthase (NOS) especially NOS1 expression in colorectal cancer (CRC) cells. More importantly, our results suggested that selective inhibition of NOS1 with its specific inhibitor Nω-Propyl-L-arginine is a suitable therapeutic strategy for hypercholesterolemia-related CRC with both efficacy and toxicity reduction. CONCLUSIONS: Our findings established that hypercholesterolemia induces the oxidant stress-dependent induction of hypoxia signaling to transcriptionally up-regulate NOS1 expression in CRC cells, and the clinically applicable NOS1 inhibitor Nω-Propyl-L-arginine represents an effective therapeutic strategy for hypercholesterolemia-related CRC.

The role of nitric oxide and hormone signaling in chronic stress, anxiety, depression and post-traumatic stress disorder.

This paper provides a summary of the role of nitric oxide (NO) and hormones in the development of chronic stress, anxiety, depression, and post-traumatic stress disorder (PTSD). These mental health conditions are prevalent globally and involve complex molecular interactions. Although there is a significant amount of research and therapeutic options available, the underlying mechanisms of these disorders are still not fully understood. The primary pathophysiologic processes involved in chronic stress, anxiety, depression, and PTSD include dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, the intracellular influence of neuronal nitric oxide synthase (nNOS) on transcription factors, an inflammatory response with the formation of nitrergic oxidative species, and reduced serotonergic transmission in the dorsal raphe nucleus. Despite the extensive literature on this topic, there is a great need for further research to clarify the complexities inherent in these pathways, with the primary aim of improving psychiatric care.

Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions.

The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.

Nitric Oxide Mitigates the Deleterious Effects Caused by Infection of Pseudomonas syringae pv. syringae and Modulates the Carbon Assimilation Process in Sweet Cherry under Water Stress.

Bacterial canker is an important disease of sweet cherry plants mainly caused by Pseudomonas syringae pv. syringae (Pss). Water deficit profoundly impairs the yield of this crop. Nitric oxide (NO) is a molecule that plays an important role in the plant defense mechanisms. To evaluate the protection exerted by NO against Pss infection under normal or water-restricted conditions, sodium nitroprusside (SNP), a NO donor, was applied to sweet cherry plants cv. Lapins, before they were exposed to Pss infection under normal or water-restricted conditions throughout two seasons. Well-watered plants treated with exogenous NO presented a lower susceptibility to Pss. A lower susceptibility to Pss was also induced in plants by water stress and this effect was increased when water stress was accompanied by exogenous NO. The lower susceptibility to Pss induced either by exogenous NO or water stress was accompanied by a decrease in the internal bacterial population. In well-watered plants, exogenous NO increased the stomatal conductance and the net CO2 assimilation. In water-stressed plants, NO induced an increase in the leaf membranes stability and proline content, but not an increase in the CO2 assimilation or the stomatal conductance.

Assessment of Anticancer Properties of Argemone mexicana L. and Berberine: A Comparative Study.

Argemone mexicana L. has been used in traditional Mexican medicine. Among its bioactive constituents, berberine (BER) has garnered attention for its cytotoxic properties against different tumor cell lines. This study investigates the in vitro toxicity against HEP-G2 (human hepatocellular carcinoma) and murine lymphoma (L5178Y-R) cells using the MTT assay of the methanol extract (AmexM), sub-partitions of A. mexicana, and BER. Selectivity indices (SIs) were determined by comparing their cytotoxic effects on VERO (monkey kidney epithelial) and PBMC (human peripheral blood mononuclear) non-tumoral cells. Additionally, the anti-hemolytic effect of these treatments was assessed using the AAPH method. The treatment with the most promising activity against tumor cells and anti-hemolytic efficacy underwent further evaluation for toxicity in Artemia salina and antioxidant activities using DPPH, ABTS, and FRAP assays. BER demonstrated an IC50 = 56.86 µg/mL in HEP-G2 cells and IC50 < 5.0 µg/mL in L5178Y-R cells, with SI values of 15.97 and >5.40 in VERO and PBMC cells, respectively. No significant hemolytic effects were observed, although AmexM and BER exhibited the highest anti-hemolytic activity. BER also demonstrated superior antioxidant efficacy, with lower toxicity in A. salina nauplii compared to the control. Additionally, BER significantly attenuated nitric oxide production. This study highlights the antiproliferative effects of A. mexicana, particularly BER, against HEP-G2 and L5178Y-R tumor cell lines, along with its selectivity towards normal cells. Furthermore, its anti-hemolytic and antioxidant potentials were demonstrated, suggesting that BER is a promising candidate for potent chemotherapeutic agents.

Impact of Inducible Nitric Oxide Synthase Activation on Endothelial Behavior under Magnesium Deficiency.

Endothelial dysfunction is a crucial event in the early pathogenesis of cardiovascular diseases and is linked to magnesium (Mg) deficiency. Indeed, in endothelial cells, low Mg levels promote the acquisition of a pro-inflammatory and pro-atherogenic phenotype. This paper investigates the mechanisms by which Mg deficiency promotes oxidative stress and affects endothelial behavior in human umbilical vascular endothelial cells (HUVECs). Our data show that low Mg levels trigger oxidative stress initially by increasing NAPDH oxidase activity and then by upregulating the pro-oxidant thioredoxin-interacting protein TXNIP. The overproduction of reactive oxygen species (ROS) activates NF-κB, leading to its increased binding to the inducible nitric oxide synthase (iNOS) promoter, with the consequent increase in iNOS expression. The increased levels of nitric oxide (NO) generated by upregulated iNOS contribute to disrupting endothelial cell function by inhibiting growth and increasing permeability. In conclusion, we provide evidence that multiple mechanisms contribute to generate a pro-oxidant state under low-Mg conditions, ultimately affecting endothelial physiology. These data add support to the notion that adequate Mg levels play a significant role in preserving cardiovascular health and may suggest new approaches to prevent or manage cardiovascular diseases.

Nitric oxide-dependent cell death in glioblastoma and squamous cell carcinoma via prodeath mitochondrial clustering.

Besides the fission-fusion dynamics, the cellular distribution of mitochondria has recently emerged as a critical biological parameter in regulating mitochondrial function and cell survival. We previously found that mitochondrial clustering on the nuclear periphery, or monopolar perinuclear mitochondrial clustering (MPMC), accompanies the anticancer activity of air plasma-activated medium (APAM) against glioblastoma and human squamous cell carcinoma, which is closely associated with oxidant-dependent tubulin remodeling and mitochondrial fragmentation. Accordingly, this study investigated the regulatory roles of nitric oxide (NO) in the anticancer activity of APAM. Time-lapse analysis revealed a time-dependent increase in NO accompanied by MPMC. In contrast, APAM caused minimal increases in MPMC and NO levels in nontransformed cells. NO, hydroxyl radicals, and lipid peroxide levels increased near the damaged nuclear periphery, possibly within mitochondria. NO scavenging prevented tubulin remodeling, MPMC, perinuclear oxidant production, nuclear damage, and cell death. Conversely, synthetic NO donors augmented all the prodeath events and acted synergistically with APAM. Salinomycin, an emerging drug against multidrug-resistant cancers, had similar NO-dependent effects. These results suggest that APAM and salinomycin induce NO-dependent cell death, where MPMC and oxidative mitochondria play critical roles. Our findings encourage further investigations on MPMC as a potential target for NO-driven anticancer agents against drug-resistant cancers.

Relationships between nitric oxide biomarkers and physiological outcomes following dietary nitrate supplementation.

Dietary nitrate (NO3-) supplementation can increase nitric oxide (NO) bioavailability, reduce blood pressure (BP) and improve muscle contractile function in humans. Plasma nitrite concentration (plasma [NO2-]) is the most oft-used biomarker of NO bioavailability. However, it is unclear which of several NO biomarkers (NO3-, NO2-, S-nitrosothiols (RSNOs)) in plasma, whole blood (WB), red blood cells (RBC) and skeletal muscle correlate with the physiological effects of acute and chronic dietary NO3- supplementation. Using a randomized, double-blind, crossover design, 12 participants (9 males) consumed NO3--rich beetroot juice (BR) (∼12.8 mmol NO3-) and NO3--depleted placebo beetroot juice (PL) acutely and then chronically (for two weeks). Biological samples were collected, resting BP was assessed, and 10 maximal voluntary isometric contractions of the knee extensors were performed at 2.5-3.5 h following supplement ingestion on day 1 and day 14. Diastolic BP was significantly lower in BR (-2 ± 3 mmHg, P = 0.03) compared to PL following acute supplementation, while the absolute rate of torque development (RTD) was significantly greater in BR at 0-30 ms (39 ± 57 N m s-1, P = 0.03) and 0-50 ms (79 ± 99 N m s-1, P = 0.02) compared to PL following two weeks supplementation. Greater WB [RSNOs] rather than plasma [NO2-] was correlated with lower diastolic BP (r = -0.68, P = 0.02) in BR compared to PL following acute supplementation, while greater skeletal muscle [NO3-] was correlated with greater RTD at 0-30 ms (r = 0.64, P=0.03) in BR compared to PL following chronic supplementation. We conclude that [RSNOs] in blood, and [NO3-] in skeletal muscle, are relevant biomarkers of NO bioavailability which are related to the reduction of BP and the enhanced muscle contractile function following dietary NO3- ingestion in humans.

The tissue-specific nature of physiological zebrafish mitochondrial bioenergetics.

Zebrafish are a powerful tool to study a myriad of experimental conditions, including mitochondrial bioenergetics. Considering that mitochondria are different in many aspects depending on the tissue evaluated, in the zebrafish model there is still a lack of this investigation. Especially for juvenile zebrafish. In the present study, we examined whether different tissues from zebrafish juveniles show mitochondrial density- and tissue-specificity comparing brain, liver, heart, and skeletal muscle (SM). The liver and brain complex IV showed the highest O2 consumption of all ETC in all tissues (10x when compared to other respiratory complexes). The liver showed a higher potential for ROS generation. In this way, the brain and liver showed more susceptibility to O2- generation when compared to other tissues. Regarding Ca2+ transport, the brain showed greater capacity for Ca2+ uptake and the liver presented low Ca2+ uptake capacity. The liver and brain were more susceptible to producing NO. The enzymes SOD and Catalase showed high activity in the brain, whereas GPx showed higher activity in the liver and CS in the SM. TEM reveals, as expected, a physiological diverse mitochondrial morphology. The essential differences between zebrafish tissues investigated probably reflect how the mitochondria play a diverse role in systemic homeostasis. This feature may not be limited to normal metabolic functions but also to stress conditions. In summary, mitochondrial bioenergetics in zebrafish juvenile permeabilized tissues showed a tissue-specificity and a useful tool to investigate conditions of redox system imbalance, mainly in the liver and brain.