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Background: Pre-eclampsia (PE) is a common condition in pregnancy; however, methods for early diagnosis and effective treatment options are lacking. Ferroptosis is a newly identified iron-dependent cell death pathway. The aim of this study was to investigate the role of ferroptosis-related genes in PE, the underlying mechanism, and their potential diagnostic value using a bioinformatics approach., Methods: We downloaded the GSE48424 and GSE98224 datasets from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between PE and healthy pregnancy samples were identified in the GSE48424 dataset and subjected to weighted gene co-expression network analysis; the most relevant modules were intersected with known ferroptosis-related genes to distinctly identify the role of ferroptosis in PE. We further searched transcription factors and microRNAs that are predicted to regulate these ferroptosis-related genes, and patients in the GSE48424 dataset were divided into two groups according to high or low expression of the key ferroptosis-related genes associated with PE. To obtain robust key ferroptosis-related genes in PE, we validated their expression levels in the external dataset GSE98224. Finally, the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay was utilized to access the expression of these genes in the PE and normal blood samples., Results: Six ferroptosis-related genes involved in PE were obtained by overlapping 3661 genes most associated with PE, 565 DEGs between PE and normal samples, and 259 known ferroptosis-related genes. Among these genes, patients with PE displaying lower expression levels of NOS2 and higher expression levels of PTGS2 had a higher ferroptosis potential index. The expression pattern of NOS2 was consistent in the GSE48424 and GSE98224 datasets. RT-qPCR data confirmed that NOS2 expression was more significantly elevated in patients with PE than in those with a normal pregnancy., Conclusions: Our study explored the diagnostic value of ferroptosis-related genes in PE, and identified NOS2 as the key gene linking ferroptosis and PE, suggesting a new candidate biomarker for early PE diagnosis., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Previous studies have indicated that nitric oxide synthase 2 ( NOS2 ) genetic variations are involved in delayed fracture healing and fracture non-union. Whether these genetic variants associate with the development of osteomyelitis (OM) remains unclear. Here, we analyzed the potential relationships between NOS2 genetic variations and the risk of developing post-traumatic OM (PTOM) in a Chinese Han population., Methods: Altogether 704 participants, including 336 PTOM patients and 368 healthy controls, were genotyped of rs2297514 and rs2248814 of the NOS2 gene using the SNaPshot genotyping method., Results: Outcomes showed that the frequency of allele C of rs2297514 in the patient group was significantly lower than that in the control group (48.7% vs . 54.5%, P = 0.029, OR = 0.792, 95% CI 0.642 - 0.976). In addition, significant associations were found between rs2297514 and susceptibility to PTOM by the recessive model ( P = 0.007, OR = 0.633, 95% CI 0.453 - 0.884), and the homozygous model ( P = 0.039, OR = 0.648, 95% CI 0.429 - 0.979). Moreover, patients with the CC genotype of rs2297514 had lower inflammatory biomarkers levels than the TT genotype, especially for the C-reactive protein (CRP) level (median: 4.1 mg/L vs . 8.9 mg/L, P = 0.027). However, no significant relationship was noted between rs2248814 and the risk of developing PTOM., Conclusion: In this Chinese cohort, rs2297514 is correlated with a decreased risk of PTOM development, with genotype CC as a protective factor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Song, Zhang, Lin, Hu, Pan, Jiang and Hu.)

Background: Multiple sclerosis (MS) is characterized by neuroinflammation and demyelination orchestrated by activated neuroglial cells, CNS infiltrating leukocytes, and their reciprocal interactions through inflammatory signals. An inflammatory stimulus triggers inducible nitric oxide synthase (NOS2), a pro-inflammatory marker of microglia/macrophages (MG/Mφ) to catalyze sustained nitric oxide production. NOS2 during neuroinflammation, has been associated with MS disease pathology; however, studies dissecting its role in demyelination are limited. We studied the role of NOS2 in a recombinant β-coronavirus-MHV-RSA59 induced neuroinflammation, an experimental animal model mimicking the pathological hallmarks of MS: neuroinflammatory demyelination and axonal degeneration., Objective: Understanding the role of NOS2 in murine-β-coronavirus-MHV-RSA59 demyelination., Methods: Brain and spinal cords from mock and RSA59 infected 4-5-week-old MHV-free C57BL/6 mice (WT) and NOS2-/- mice were harvested at different disease phases post infection (p.i.) (day 5/6-acute, day 9/10-acute-adaptive and day 30-chronic phase) and compared for pathological outcomes., Results: NOS2 was upregulated at the acute phase of RSA59-induced disease in WT mice and its deficiency resulted in severe disease and reduced survival at the acute-adaptive transition phase. Low survival in NOS2-/- mice was attributed to (i) high neuroinflammation resulting from increased accumulation of macrophages and neutrophils and (ii) Iba1 + phagocytic MG/Mφ mediated-early demyelination as observed at this phase. The phagocytic phenotype of CNS MG/Mφ was confirmed by significantly higher mRNA transcripts of phagocyte markers-CD206, TREM2, and Arg1 and double immunolabelling of Iba1 with MBP and PLP. Further, NOS2 deficiency led to exacerbated demyelination at the chronic phase as well., Conclusion: Taken together the results imply that the immune system failed to control the disease progression in the absence of NOS2. Thus, our observations highlight a protective role of NOS2 in murine-β-coronavirus induced demyelination., (© 2023. The Author(s).)

Studies of mouse models of Alzheimer's disease (AD) have demonstrated that nitric oxide synthase 2 (NOS2) is involved in AD pathology. However, the effects of NOS2 on the pathology of Parkinson's disease (PD) are not well studied. To address this gap, we examined the impact of NOS2 on disease-associated phenotypes in a mouse model of PD. Transgenic mice carrying the A53T mutation of α-synuclein (Syn A53T ) and newly generated double transgenic mice with deletion of NOS2 (Syn A53T /NOS2 -/- ) were used. Compared with Syn A53T mice, the loss of nos2 decreased α-synuclein phosphorylation at serine 129 and reduced α-synuclein-induced microglial and astrocyte activation in Syn A53T /NOS -/- mice. Additionally, neuroinflammation-related gene clusters in the deep mesencephalic nucleus (DpMe) were altered in Syn A53T /NOS -/- mice compared with Syn A53T mice. Taken together, our results suggest that deletion of nos2 alleviates α-synuclein pathology and α-synuclein-associated neuroinflammatory responses in the brain., (© 2023. The Author(s).)

Background and Objectives : In paediatric population, atopic asthma is associated with increased eosinophil counts in patients, that correlate with the airway inflammation measured by the concentration of nitric oxide in exhaled air (FeNO). As the FeNO level is a biomarker of atopic asthma, we assumed that polymorphisms in nitric synthases genes may represent a risk factor for asthma development. The purpose of this study was to analyse the association of NOS genetic variants with childhood asthma in the Polish population. Materials and methods : In study we included 443 children-220 patients diagnosed with atopic asthma and 223 healthy control subjects. We have genotyped 4 single nucleotide polymorphisms (SNP) from 3 genes involved in the nitric oxide synthesis ( NOS1 , NOS2 and NOS3 ). All analyses were performed using polymerase chain reaction with restriction fragments length polymorphism (PCR-RFLP). Results: We observed significant differences between cases and controls in SNP rs10459953 in NOS2 gene, considering both genotypes ( p = 0.001) and alleles ( p = 0.0006). The other analyzed polymorphisms did not show association with disease. Conclusions : According to our results, 5'UTR variant within NOS2 isoform may have an impact of asthma susceptibility in the population of Polish children. Further functional studies are required to understand the role of iNOS polymorphism in NOS2 translation and to consider it as a novel risk factor in childhood asthma. The next step would be to apply this knowledge to improve diagnosis and develop novel personalized asthma therapies.

Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This study aimed to unravel the regulatory role of noncoding RNAs (ncRNA) on the nitric oxide (NO) machinery system in triple-negative breast cancer (TNBC) patients and to further assess the influence of NO-modulating ncRNAs on TNBC progression, immunogenic profile, and the tumor microenvironment (TME). The results revealed miR-939-5p and lncRNA HEIH as novel ncRNAs modulating NO machinery in TNBC. MiR-939-5p, an underexpressed microRNA (miRNA) in BC patients, showed an inhibitory effect on NOS2 and NOS3 transcript levels on TNBC cells. In contrast, HEIH was found to be markedly upregulated in TNBC patients and showed a modulatory role on miR-939-5p/NOS2/NO axis. Functionally, miR-939-5p was characterized as a tumor suppressor miRNA while HEIH was categorized as a novel oncogenic lncRNA in TNBC. Finally, knocking down of HEIH resulted in improvement of immunogenic profile of TNBC cells through inducing MICA/B and suppressing the immune checkpoint inhibitor PDL1. In the same context, knockdown of HEIH resulted in the alleviation of the immune-suppressive TME by repressing interleukin-10 and tumor necrosis factor-α levels. In conclusion, this study identifies miR-939-5p as a tumor suppressor miRNA while HEIH as an oncogenic lncRNA exhibiting its effect through miR-939-5p/NOS2/NO axis. Therefore, repressing BC hallmarks, improving TNBC immunogenic profile, and trimming TME., (© 2020 Wiley Periodicals LLC.)

Patients with tension-type headache (TTH) have an increased risk of developing arterial hypertension (AH), while hypertensive subjects do seem to have an increased risk of TTH. We searched for full-text English publications in databases using keywords and combined word searches over the past 15 years. In addition, earlier publications of historical interest were included in the review. In our review, we summed up the single nucleotide variants (SNVs) of Nitric Oxide Synthases ( NOSs ) genes involved in the development of essential AH and TTH. The results of studies we discussed in this review are contradictory. This might be due to different designs of the studies, small sample sizes in some of them, as well as different social and geographical characteristics. However, the contribution of genetic and environmental factors remains understudied. This makes the issue interesting for researchers, as understanding these mechanisms can contribute to a search for new approaches to pathogenetic and disease-modifying treatment of the AH and TTH phenotype. New drugs against AH and TTH can be based on inhibition of nitric oxide (NO) production, blockade of steps in the NO-cGMP pathway, or NO scavenging. Indeed, selective neuronal NOS (n-NOS) and inducible NOS (i-NOS) inhibitors are already in early clinical development.

Purpose: We investigated N w -hydroxy l-Arginine (NOHA) predictive response in serous ovarian carcinoma based on estrogen-hormone receptor expression status; and assessed the distinctive NOHA response between estrogen-receptor-negative (ER-) tumor subtypes of ovarian and breast cancer., Materials/methods: Three-dimensional (3D) spheroids models of ER- and estrogen-receptor-positive (ER+) from breast and ovarian tumor, cultured for 9 weeks, were assayed for cellular levels of inducible nitric oxide synthase (NOS2), nitric oxide (as total nitrite) and l-Arginine, and compared to NOHA in culture medium. Statistical difference was set at p < 0.01., Results: Nine-week in vitro studies showed a progressive NOHA reduction in culture medium by at least 0.4-0.8 fold, and 0.65-0.92 fold only in the ER- breast tumor and ER- ovarian tumor 3D spheroids, respectively; with increases in cellular NOS2 and nitric-oxide levels, by at least 1.0-2.45 fold in both ER- tumor subtype 3D spheroids (p < 0.01; n = 6). Within ER- subtypes, medium NOHA decreased by ≥ 38.9% in ovarian cancer over breast cancer 3D-spheroids, with cellular increases in NOS2 (by ≥ 17.4%), and nitric oxide (by ≥ 18.8%). Cellular l-Arginine to medium NOHA ratio was higher, and by at least 6.5-22.5 fold in ER- breast tumor 3D-spheroids, and at least 10-70 fold in ER- ovarian tumor 3D spheroids, than in ER+ and control conditions; and was ≥48% higher in ER- ovarian cancer than in ER- breast cancer 3D-spheroids., Conclusions: The present study shows NOHA as a sensitive and selective indicator differentiating and distinguishing ER- subtypes based on the tumor grade., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

Background: Increasing evidence has suggested that a single nucleotide polymorphism in the Ncf1 gene is associated with experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of NCF1-induced immunoregulatory effects remain poorly understood. In this study, we focus on NCF1 deficiency-mediated effects on EAE in NOS2 dependent and independent ways., Methods: To determine the effects of NCF1 and NOS2 during EAE development, we have established recombinant mouse strains deficient at NCF1 and/or NOS2 in a crossbreeding system. Different strains allow us to examine the entire course of the disease in the Nos2-null mice bearing a Ncf1 gene that encodes a mutated NCF1, deficient in triggering oxidative burst, after immunization with recombinant myelin oligodendrocyte glycoprotein (MOG) 79-96 peptides. The peptide-induced innate and adaptive immune responses were analyzed by flow cytometry., Results: NCF1-deficient mice developed a reduced susceptibility to EAE, whereas NCF1-NOS2 double-deficient mice developed an enhanced EAE, as compared with NOS2-deficient mice. Flow cytometry analyses show that double deficiencies resulted in an increase of neutrophils in the spleen, accompanied with higher release of interleukin-1β in neutrophils prior to EAE onset. The additional deficiency in NCF1 had no added effect on either interleukin-17 or interferon-γ secretion of T cells during the priming phase., Conclusions: These studies show that NCF1 and NOS2 interact to regulate peptide-induced EAE.

The role of nitric oxide (NO) in cancer progression has largely been studied in the context of tumor NOS2 expression. However, pro- versus anti-tumor signaling is also affected by tumor cell-macrophage interactions. While these cell-cell interactions are partly regulated by NO, the functional effects of NO flux on proinflammatory (M1) macrophages are unknown. Using a triple negative murine breast cancer model, we explored the potential role of macrophage Nos2 on 4T1 tumor progression. The effects of NO on macrophage phenotype were examined in bone marrow derived macrophages from wild type and Nos2 -/- mice following in vitro stimulation with cytokine/LPS combinations to produce low, medium, and high NO flux. Remarkably, Nos2 induction was spatially distinct, where Nos2 high cells expressed low cyclooxygenase-2 (Cox2) and vice versa. Importantly, in vitro M1 polarization with IFNγ+LPS induced high NO flux that was restricted to cells harboring depolarized mitochondria. This flux altered the magnitude and spatial extent of hypoxic gradients. Metabolic and single cell analyses demonstrated that single cell Nos2 induction limited the generation of hypoxic gradients in vitro, and Nos2-dependent and independent features may collaborate to regulate M1 functionality. It was found that Cox2 expression was important for Nos2 high cells to maintain NO tolerance. Furthermore, Nos2 and Cox2 expression in 4T1 mouse tumors was spatially orthogonal forming distinct cellular neighborhoods. In summary, the location and type of Nos2 high cells, NO flux, and the inflammatory status of other cells, such as Cox2 high cells in the tumor niche contribute to Nos2 inflammatory mechanisms that promote disease progression of 4T1 tumors., (Published by Elsevier B.V.)

A growing body of evidence supports the use of probiotics in the treatment of several skin conditions, including wounds. Even if in vitro and in vivo studies have highlighted the pro-healing effects of some probiotic bacteria, the underlying mechanisms are still not fully defined. The current investigation aimed to determine the re-epithelialization potential of the soluble fraction from lysate of seven different probiotic strains belonging to different genera (i.e., Streptococcus , Lactobacillus , and Bifidobacterium ) on in vitro physically wounded HaCaT monolayer model. The results suggested that the soluble fraction of S. thermophilus, L. plantarum , and L. acidophilus promoted the re-epithelialization of scratched HaCaT monolayers, whereas those from B. longum, B. infantis showed no significant effect on in vitro wound repair. The mechanisms underlying the pro- or anti-healing properties of selected bacterial strains strictly and positively correlated with their ability to modulate nitric oxide synthase 2 (NOS2) expression and activity. Accordingly, the pre-treatment with aminoguanidine (AG), a specific inhibitor of NOS2 activity, abrogated the pro-healing effects of B. breve significantly inhibited the process. On the other hand, L. bulgaricus showed no significant effect on in vitro wound repair. The mechanisms underlying the pro- or anti-healing properties of selected bacterial strains strictly and positively correlated with their ability to modulate nitric oxide synthase 2 (NOS2) expression and activity. Accordingly, the pre-treatment with aminoguanidine (AG), a specific inhibitor of NOS2 activity, abrogated the pro-healing effects of S. thermophilus, L. plantarum , and L. acidophilus ., Competing Interests: The authors declare no conflict of interest.

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

Background/aims: Type 1 Diabetes (T1D) involves autoimmune attack due to reduced regulatory T cells as an effect of mutant Stat5b(C1462A) in non-obese diabetic (NOD) mice, a T1D model resulting in pancreatic β-cell destruction. Although reactive oxygen species are considered to orchestrate the immune attack, the role of nitric oxide (·NO) still remains debatable. Since JAK-STAT pathway is known to induce Nos2, we investigated the role of STAT5B in nitric oxide generation and oxidative stress., Methods: In this study, we have used chromatin immunoprecipitation with STAT5B antibody to explore whether STAT5B binds Nos2 promoter. Using Stat5b gene silencing and overexpression models in MIN6 mouse pancreatic β-cell line we have assayed nitric oxide and its end products, superoxide levels, H₂O₂ levels, and expression of genes related to redox pathway by immunocytochemistry, biochemical assays, quantitative real time PCR and western blotting., Results: Our results prove that STAT5B binds to the candidate gamma-interferon-activated (GAS) element in Nos2 promoter thereby inducing Nos2 mRNA transcription resulting in NOS2 protein expression in MIN6, a mouse pancreatic β-cell line. Our findings are substantiated by reduced ·NO as well as nitric oxide end products (nitrate and nitrite), and increased superoxide production in Stat5b silenced MIN6 cells. Our results indicate that C1462A mutant STAT5B shows lack of ·NO generation ability. To detoxify excess superoxide as a consequence of lowered Nos2, an overexpressed SOD2 in Stat5b silenced cells results in increased H₂O₂ production. H₂O₂ metabolizing enzymes do not show upregulation upon Stat5b silencing, and thus oxidative stress is brought about by amassed H₂O₂. Stat5b silencing finally reduces AKT expression, a prosurvival signal., Conclusion: Our study enables us to conclude that β-cell stress is aggravated by the incapability of STAT5B to induce Nos2 resulting in H₂O₂ accumulation and the ensuing oxidative stress enhances β-cell damage., Competing Interests: The authors declare no competing financial interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Aberrant nitric oxide synthase 2 (NOS2) expression has been suggested as an interesting therapeutic target that is being implicated as a component of the molecular profile of several human malignant tumors, including glioblastoma, which is the most aggressive brain tumor with limited therapeutic options and poor prognosis. The aim of the present work was to evaluate the effect of 1400W, a specific NOS2 inhibitor, on human glioma cells in terms of clonogenic potential, proliferation, migration rate, and neurosphere generation ability. NOS2 expression was determined by Western blotting. Nitric oxide (NO) production was measured through nitrite level determination. The trypan blue exclusion test and the plate colony formation assay were performed to evaluate cell proliferation and clonogenic potential. Cell proliferation and migration ability was assessed by the in vitro wound-healing assay. Neurosphere generation in a specific stemcell medium was investigated. NOS2 was confirmed to be expressed in both the glioma cell line and a human glioma primary culture, and overexpressed in relative derived neurospheres. Experiments that aimed to evaluate the influence of 1400W on U-87 MG, T98G (glioblastoma cell lines) and primary glioma cells sustained the crucial role played by NOS2 in proliferation, colony formation, migration, and neurosphere generation, thus supporting the emerging relevance of a NOS2/NO system as a prognostic factor for glioma malignancy and recurrence.

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER - ) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER - patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFα-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER - and TNBC disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Leptin, following its discovery, has developed a formidable interest in the scientific community to delineate its contribution towards overall metabolic homeostasis. Contradictory reports have been published on leptin administration effects on whole body insulin sensitivity. Following late reports, we surveyed human serum leptin levels along with other metabolic parameters including BMI and HOMA-IR. We found a positive correlation between leptin levels and insulin resistance parameters. Considering the presence of the long form of leptin receptor on adipocytes, we explored the effects of chronic physiological hyper-leptinemic exposure on adipocyte insulin sensitivity. Chronic leptin (50ng/ml) treatment in 3T3-L1 adipocytes decreased insulin-induced phosphorylation of nodal insulin signaling proteins along with reduced glucose uptake. Metabolic flux studies indicated mitochondrial dysfunction and reduced oxygen consumption rate. Leptin treatment also increased both cellular and mitochondrial superoxide levels concomitant to increased expression of nitric oxide synthase-2 (NOS2). Further, pharmacological depletion of NOS2 reversed leptin mediated effects on insulin signaling. In-vivo implantation of leptin osmotic pumps in C57BL/6 mice also decreased insulin responsiveness. Interestingly, these effects were lacking in NOS2 knockout strain. In conclusion, our studies put forward a potential link between leptin and adipocyte insulin responsiveness in an NOS2 dependent manner., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background/aim: To clarify the mechanisms responsible for the anti-inflammatory/proinflammatory activities of eugenol-related compounds, we investigated the cytotoxicity and up-regulatory/down-refgulatory effects of the biphenols curcumin, bis-eugenol, magnolol and honokiol, and the monophenols eugenol and isoeugenol, on major regulators of cyclooxygenase-2 (Cox-2), nitric oxide synthase 2 (Nos2) and heme oxygenase-1 (HO-1) mRNA in RAW264.7 cells., Materials and Methods: mRNA expression was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and the theoretical parameters were calculated using the DFT/B3LYP/6-31* method. Also, the antioxidant activity of eugenol-related compounds in combination with 2-mercapto-1-methylimidazole (MMI, as a model for glutathione (GSH)) was investigated using the induction period method for polymerization of methyl methacrylate initiated by benzoyl peroxide (BPO)., Results: The cytotoxicity of eugenol-related compounds showed a linear relationship with their softness (σ) and electrophilicity (ω). At a concentration of 50 μM, biphenols except for bis-eugenol elicited the expression of mRNA for both Cox-2 and Nos2, but monophenols did not. In contrast, bis-eugenol elicited Cox-2 gene expression, but down-regulated Nos2 gene expression. bis-Eugenol alone induced the expression of HO-1 mRNA, and when combined with MMI it showed a potent antagonistic effect on BPO-induced antioxidant activity. The ability of methoxyphenols to inhibit LPS-stimulated Cox-2 gene expression declined in the order curcumin >> isoeugenol > bis-eugenol >> eugenol, and the rank of ability was related to their ω value., Conclusion: Most eugenol-related compounds had proinflammatory activity at high concentrations. However, they had also anti-inflammatory activity at lower concentrations. Eugenol-related compounds may exert antioxidant and anti-inflammatory activity in LPS-stimulated RAW264.7 cells possibly by inhibiting the activation of nuclear factor-kappa B (Nf-ĸB), whereas bis-eugenol requires induction of HO-1 expression. bis-Eugenol as well as curcumin, may have anti-inflammatory and anticancer therapeutic applications., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The pathogen Mycobacterium leprae causes leprosy that affects mainly skin and nerves. Polymorphisms of certain genes are substantiated to be associated with the susceptibility/resistance to leprosy. The present investigation addressed the association of Nitric Oxide Synthase2 gene polymorphisms and leprosy in a population from northern part of India. A total of 323 leprosy cases and 288 healthy controls were genotyped for four NOS2 promoter variants (rs1800482, rs2779249, rs8078340 and rs2301369) using FRET technology in Real Time PCR. None of these SNPs in promoter sites was associated with susceptibility/resistance to leprosy. NOS2 rs1800482 was found to be monomorphic with GG genotype. However, NOS2-1026T allele was observed to be in higher frequency with leprosy cases (BL and LL) who were not suffering from any reactional episodes compared to cases with ENL reaction {OR=0.30, 95% CI (0.10-0.86), p=0.024}. NOS2-1026GT genotype was more prevalent in cases without reaction (BT, BB and BL) compared to RR reactional patients {OR=0.38, 95% CI (0.17-0.86), p=0.02}. Although haplotype analysis revealed that no haplotype was associated with leprosy susceptibility/resistance with statistical significance, GTG haplotype was noted to be more frequent in healthy controls. These SNPs are observed to be in linkage disequilibrium. Although, these SNPs are not likely to influence leprosy vulnerability, -1026G>T SNP was indicated to have noteworthy role in leprosy reactions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Significance: Breast cancer is the second leading cause of cancer-related deaths among women in the United States. Development and progression of malignancy are associated with diverse cell signaling pathways that control cell proliferation, survival, motility, invasion, and metastasis. Recent Advances: An increasing number of clinical studies have implicated a strong relationship between elevated tumor nitric oxide synthase-2 (NOS2) expression and poor patient survival., Critical Issues: Herein, we review what we believe to be key mechanisms in the role(s) of NOS2-derived nitric oxide (NO) as a driver of breast cancer disease progression. High NO increases cyclooxygenase-2 activity, hypoxia inducible factor-1 alpha protein stabilization, and activation of important cell signaling pathways, including phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinase, epidermal growth factor receptor, and Ras, through post-translational protein modifications. Moreover, dysregulated NO flux within the tumor microenvironment has other important roles, including the promotion of angiogenesis and modulation of matrix metalloproteinase/tissue inhibitor matrix metalloproteinase associated with tumor progression., Future Directions: The elucidation of these and other NO-driven pathways implicates NOS2 as a key driver of breast cancer disease progression and provides a new perspective in the identification of novel targets that may be therapeutically beneficial in the treatment of estrogen receptor-negative disease. Antioxid. Redox Signal. 26, 1044-1058.

Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

Novel specific therapies for psoriasis and eczema have been developed, and they mark a new era in the treatment of these complex inflammatory skin diseases. However, within their broad clinical spectrum, psoriasis and eczema phenotypes overlap making an accurate diagnosis impossible in special cases, not to speak about predicting the clinical outcome of an individual patient. Here, we present a novel robust molecular classifier (MC) consisting of NOS2 and CCL27 gene that diagnosed psoriasis and eczema with a sensitivity and specificity of >95% in a cohort of 129 patients suffering from (i) classical forms; (ii) subtypes; and (iii) clinically and histologically indistinct variants of psoriasis and eczema. NOS2 and CCL27 correlated with clinical and histological hallmarks of psoriasis and eczema in a mutually antagonistic way, thus highlighting their biological relevance. In line with this, the MC could be transferred to the level of immunofluorescence stainings for iNOS and CCL27 protein on paraffin-embedded sections, where patients were diagnosed with sensitivity and specificity >88%. Our MC proved superiority over current gold standard methods to distinguish psoriasis and eczema and may therefore build the basis for molecular diagnosis of chronic inflammatory skin diseases required to establish personalized medicine in the field., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pancreatic cancer is one of the most lethal malignancies and is refractory to the available treatments. Pancreatic ductal adenocarcinoma (PDAC) expresses high level of inducible nitric oxide synthase (NOS2), which causes sustained production of nitric oxide (NO). We tested the hypothesis that an aberrantly increased NO-release enhances the development and progression of PDAC. Enhanced NOS2 expression in tumors significantly associated with poor survival in PDAC patients (N = 107) with validation in independent cohorts. We then genetically targeted NOS2 in an autochthonous mouse model of PDAC to examine the effect of NOS2-deficiency on disease progression and survival. Genetic ablation of NOS2 significantly prolonged survival and reduced tumor severity in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice. Primary tumor cells isolated from NOS2-deficient KPC (NKPC) mice showed decreased proliferation and invasiveness as compared to those from KPC mice. Furthermore, NKPC tumors showed reduced expression of pERK, a diminished inactivation of Forkhead box transcription factor O (FOXO3), a tumor suppressor, and a decrease in the expression of oncomir-21, when compared with tumors in KPC mice. Taken together, these findings showed that NOS2 is a predictor of prognosis in early stage, resected PDAC patients, and provide proof-of-principle that targeting NOS2 may have potential therapeutic value in this lethal malignancy., Competing Interests: None.

Background: Harmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to the upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca(2+)-sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca(2+) influx, we hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such as Nos2., Methods: We studied microglia in vivo and in primary culture from adult rats, and from wild type, Abcc8-/- and Trpm4-/- mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation., Results: In microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation of Nos2 mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing of Abcc8 or Trpm4 reduced Nos2 upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca(2+)]i), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca(2+)]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca(2+)/calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation., Conclusions: Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca(2+)-sensitive gene expression, including Nos2.

Compelling evidence indicates that the immune system is linked to metabolism in gestational diabetes mellitus (GDM), but factors participating in these processes still are awaiting identification. Inducible nitric oxide synthase, encoded by the NOS2 gene, and surfactant protein D, encoded by the SFTPD gene, have been implicated in diabetes. We investigated NOS2 and SFTPD mRNA levels in leukocytes obtained from 125 pregnant women with (n = 87) or without (control group; n = 38) GDM, and, in turn, correlated their expression with clinical parameters of subjects. Leukocytes were isolated from the blood of pregnant women and NOS2 and SFTPD expression in these cells was determined by quantitative real time PCR (qRT-PCR). Univariate correlation analyses were performed to assess an association between leukocyte NOS2 and SFTPD expression and clinical characteristics of patients. qRT-PCR experiments disclosed significantly increased leukocyte NOS2 and SFTPD mRNA levels in hyperglycemic GDM patients (P < 0.05). In the entire study group, there were significant positive associations of leukocyte NOS2 and SFTPD mRNAs with C-reactive protein. Additionally, transcript level of SFTPD also correlated positively with fasting glycemia and insulin resistance. This study demonstrates that an impaired glucose metabolism in GDM may be predominant predictor of leukocyte NOS2 and SFTPD overexpression in diabetic patients. Furthermore, alterations in the expression of these genes are associated with glucose metabolism dysfunction and/or inflammation during pregnancy. In addition, these findings support the utilization of leukocytes as good experimental model to study a relationship between immune-related genes and metabolic changes in women with GDM, as well as to assess the potential mechanisms underlying these alterations., (© 2015 by the Society for Experimental Biology and Medicine.)

Ovarian cancer constitutes one of the most lethal gynaecological malignancies worldwide and currently no satisfactory therapeutic approaches have been established. Therefore, elucidation of molecular mechanisms to develop targeted therapy of ovarian cancer is crucial. PDLIM2 is critical to promote ubiquitination of nuclear p65 and thus its role in inflammation has been highlighted recently. We demonstrate that PDLIM2 is decreased in both ovarian high-grade serous carcinoma and in various human ovarian cancer cell lines compared with normal ovary tissues and human ovarian surface epithelial cells (HOSE). Further functional analysis revealed that PDLIM2 is epigenetically repressed in ovarian cancer development and inhibition of PDLIM2 promoted ovarian cancer growth both in vivo and in vitro via NOS2-derived nitric oxide signaling, leading to recruitment of M2 type macrophages. These results suggest that PDLIM2 might be involved in ovarian cancer pathogenesis, which could serve as a promising therapeutic target for ovarian cancer patients.

Background: Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined., Materials and Methods: Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK., Results: The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression., Conclusions: These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1(fl/fl)) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1(fl/fl)Tie2cre). Macrophages from Gch1(fl/fl)Tie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1(fl/fl)Tie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1(fl/fl)Tie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1(fl/fl)Tie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1(fl/fl)Tie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1(fl/fl)Tie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)