ChapterLiterature Review

Immunobiology of Nitric Oxide and Regulation of Inducible Nitric Oxide Synthase

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Abstract

Nitric oxide (NO) is a bioactive gas that has multiple roles in innate and adaptive immune responses. In macrophages, nitric oxide is produced by inducible nitric oxide synthase upon microbial and cytokine stimulation. It is needed for host defense against pathogens and for immune regulation. This review will summarize the role of NO and iNOS in inflammatory and immune responses and will discuss the regulatory mechanisms that control inducible nitric oxide synthase expression and activity.

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... The iNOS gene promoter is very complex and is activated by an additive effect of various transcription factors such as AP-1, C/EBP, CREB, GATA, HIF, IRF-1, NF-AT, NF-κB, NF-IL6, Oct-1, PARP1, PEA3, p53, Sp1, SRF, STAT-1α, TBE, TCF, and YY1 (Pautz et al., 2010;Guo et al., 2016). In epithelial cells, the combined action of some cytokines, being the most important interleukin 1β (IL-1β), interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α), and/or some proinflammatory stimuli such as lipopolysaccharides (LPS) triggers the activation of the transcription factors involved in the induction of iNOS gene expression (Donnelly and Barnes, 2002;Roy et al., 2004;Guo et al., 2016;Lee et al., 2017). On the other hand, guanidinosubstituted analogs of L-arginine or methylarginines, such as asymmetric dimethylarginine (ADMA), inhibit the synthesis of NO by competing with L-arginine at the active site of NO synthases. ...
... In epithelial cells, IL-1β and TNFα stimulation induce the activation and translocation into the nucleus of nuclear transcription factor κB (NF-κB). However, INF-γ stimulation activates STAT-1 and IRF-1 (Lee et al., 2017). The synergic effect between IL-1β, TNF-α, and INF-γ is due in part to different mechanisms. ...
... However, high concentrations of NO inhibit Th1 responses by decreasing the IL-12 production of macrophages. Therefore, NO might regulate the balance between Th1 and Th2 depending on its concentration by increasing Th1 apoptosis at high concentrations and inhibiting it at low concentrations (Ibiza and Serrador, 2008;Lee et al., 2017). In contrast to this data, the addition of NO to bronchial epithelial cells showed a reduction in both Th1 and Th2 proliferation. ...
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Airway epithelium forms a physical barrier that protects the lung from the entrance of inhaled allergens, irritants, or microorganisms. This epithelial structure is maintained by tight junctions, adherens junctions and desmosomes that prevent the diffusion of soluble mediators or proteins between apical and basolateral cell surfaces. This apical junctional complex also participates in several signaling pathways involved in gene expression, cell proliferation and cell differentiation. In addition, the airway epithelium can produce chemokines and cytokines that trigger the activation of the immune response. Disruption of this complex by some inflammatory, profibrotic, and carcinogens agents can provoke epithelial barrier dysfunction that not only contributes to an increase of viral and bacterial infection, but also alters the normal function of epithelial cells provoking several lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or lung cancer, among others. While nitric oxide (NO) molecular pathway has been linked with endothelial function, less is known about the role of the NO system on the bronchial epithelium and airway epithelial cells function in physiological and different pathologic scenarios. Several data indicate that the fraction of exhaled nitric oxide (FENO) is altered in lung diseases such as asthma, COPD, lung fibrosis, and cancer among others, and that reactive oxygen species mediate uncoupling NO to promote the increase of peroxynitrite levels, thus inducing bronchial epithelial barrier dysfunction. Furthermore, iNOS and the intracellular pathway sGC-cGMP-PKG are dysregulated in bronchial epithelial cells from patients with lung inflammation, fibrosis, and malignancies which represents an attractive drug molecular target. In this review we describe in detail current knowledge of the effect of NOS-NO-GC-cGMP-PKG pathway activation and disruption in bronchial epithelial cells barrier integrity and its contribution in different lung diseases, focusing on bronchial epithelial cell permeability, inflammation, transformation, migration, apoptosis/necrosis, and proliferation, as well as the specific NO molecular pathways involved.
... In an M1 macrophage, inducible nitric oxide synthase (iNOS) is upregulated, resulting in the catabolism of arginine to citrulline and nitric oxide (II), the latter playing key role in the intracellular killing of pathogens [3]. iNOS transcription is induced synergistically through a combination of cytokines (such as IL-1β, IFNγ, TNF-α) and/or bacterial-derived products such as LPS which leads to the activation of multiple transcription factors [4]. NO produced by iNOS can cause DNA damage in the form of strand breaks and deamination of guanine and adenine [5,6]. ...
... In many circumstances, some cytotoxic effects of NO have been attributed to effects of peroxynitrite which can cause oxidation and nitrosylation of individual nucleotides [7][8][9][10]. Growth of bacterial species and malignant cells is inhibited directly by NO and this gas increases the cytotoxic activity of H 2 O 2 that is produced by activated neutrophils and macrophages [4,11]. ...
... Incubation of macrophages with IFNγ and TNF-α stimulates iNOS expression while suppressing arginase expression. Conversely, IL-4 and IL-10 upregulate arginase and suppress iNOS induction [4,14]. ...
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The method for assessing the level of nitric oxide (II) (NO) by voltammetric monitoring of nitrite ions was carried out on models M1 and M2 of polarized macrophages induced from monocytes of human peripheral blood with the addition of lipopolysaccharide (LPS) and interleukin-4 (IL-4), respectively. The model of induction of M1 and M2 macrophages was used in the work to achieve the corresponding shifts in the functional status of studied cells. Ethyl nitrite (EtONO) was used as a standard compound of nitrite ions for electrochemical measurements. Electrochemical determination of nitrite ions was performed by anodic linear sweep voltammetry in the first-order derivative mode (ALSV FOD) in Britton-Robinson (BR) buffer with pH 4.02 on carbon ink modified graphite electrode. EtONO calibrations were linear over a concentration range from 2 to 9 μmol L−1 with corresponding regression equation y = 0.768c − 0.048. Limit of detection (LOD) (S/N = 3) was 0.38 μmol L−1. The results of the study showed the fundamental possibility of using voltammetry to assess indirectly the production of nitric oxide by cells in supernatants of the monocytic macrophage lineage. The level of nitric oxide metabolites (nitrite ions) in supernatants was associated with the functional state of macrophages.
... These cells synthesize and release inflammatory mediators, including nitric oxide (NO) [1,2]. NO is a pleiotropic mediator formed from L-arginine and oxygen by three NO synthase (NOS) isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) [3]. The constitutive NOS (cNOS), nNOS and eNOS, are constantly present and synthesize small amounts of NO in response to physical or receptor stimulation. ...
... The constitutive NOS (cNOS), nNOS and eNOS, are constantly present and synthesize small amounts of NO in response to physical or receptor stimulation. In contrast, iNOS is induced by inflammatory-like stimuli and can produce large amounts of NO that predominate during inflammation [3]. eNOS-produced NO has both pro-and anti-inflammatory properties. ...
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Nitric oxide (NO) is an important part of the host defense mechanism; however, it displays both pro- and anti-inflammatory properties depending on its location and concentration. Importantly, excessive or inappropriate NO production can cause tissue damage. Systemic and local administration of NO synthase (NOS) inhibitors ameliorates and may exacerbate the inflammatory response, respectively. Here, we used a carrageenan-induced pleurisy model of acute inflammation in rats to confirm the location-dependent effects of NO and investigate the underlying mechanisms. As expected, localized suppression of NO production exacerbated inflammation, as evidenced by increased pleural exudate volumes and leukocyte counts and enhanced activity of enzymes related to oxidative stress. In contrast, local NO supplementation reduced leukocyte infiltration, vascular permeability, and the activity of oxidative stress-related enzymes. Interestingly, inhibition of heme oxygenase-1 (HO-1) reversed the anti-inflammatory effects of localized NO production, while the addition of hemin (HO-1 substrate) or carbon monoxide (CO; HO-1 metabolite) decreased leukocyte migration and exudation. Together, these findings confirm a protective role for NO at the inflammatory site, which appears to be mediated via NOS induction of the HO-1/CO pathway. Thus, NO supplementation may be a potential new treatment for oxidative stress-associated inflammatory diseases.
... Then, we conducted specific studies using peripheral blood mononuclear cells isolated from both non-sensitized and sensitized patients to Ni, respectively [35,36]. Release of interferon (IFN)-γ by T helper (h)-1 cells [37], interleukin (IL)-4, as a product of Th2 cells [38], IL-10 by T regulatory (Treg) cells [39,40], IL-17 by Th17 cells [41,42], and nitric oxide (NO) [43][44][45] was assessed in the presence of different stimuli. Results demonstrated that polyphenols were able to modulate the immune response to Ni and LPS interfering with TLR-4 binding [36]. ...
... Here we have documented that polyphenols reduce concentrations of NO in polyphenoladministered patients, thus, decreasing oxidative stress at tissue level [85]. Besides, it is well known that NO per se activates the Th1 cell subset [44,45]. ...
Article
Background Our previous findings demonstrated that in vitro supplementation of polyphenols, extracted from seeds of red grape (Nero di Troia cultivar), to peripheral lymphomonocytes from patients affected by allergic contact dermatitis (ACD) to nickel (Ni) could reduce release of pro-inflammatory cytokines and nitric oxide (NO), while increasing levels of interleukin (IL)-10, an anti-inflammatory cytokine. Objective To assess whether an intervention with oral administration of polyphenols leads to a reduction of peripheral biomarkers in ACD patients. Method At T0, 25 patients affected by ACD to Ni were orally administered with 300 mg polyphenols prodie extracted from seeds of red grape (Nero di Troia cultivar) (NATUR-OX®) for 3 months (T1). Other 25 patients affected by ACD to Ni received placebo only for the same period of time. Serum biomarkers were analyzed at T0 and T1. In both groups seven drop outs were recorded. Result At T1 in comparison to T0, in treated patients, values of IFN-γ, IL-4, IL-17, PTX3 and NO decreased, while IL-10 levels increased when compared with T0 values. Conversely, in placebo-treated patients no modifications of biomarkers were evaluated at T1. Conclusion: Present laboratory data rely on the anti-oxidant, anti-inflammatory and anti-allergic properties of polyphenols.
... Among neuronal mediators, particular attention should be directed towards nitric oxide, a neuromodulator involved in the control of vascular tone and blood pressure [61]. For example, iNOs is upregulated under stress conditions; in fact, in the presence of inflammatory cytokines and other agents (antigens of pathogens, apoptotic bodies, etc.), the expression of this enzyme increases, underlining its possible role in the inflammatory phase of wound healing, in which it could guarantee vasodilation and antibacterial activity. ...
Article
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PDT is a two-stage treatment that combines light energy with a photosensitizer designed to destroy cancerous and precancerous cells after light activation. Photosensitizers are activated by a specific wavelength of light energy, usually from a laser. The photosensitizer is nontoxic until it is activated by light. However, after light activation, the photosensitizer becomes toxic to the targeted tissue. Among sensitizers, the topical use of ALA, a natural precursor of protoporphyrin IX, a precursor of the heme group, and a powerful photosensitizing agent, represents a turning point for PDT in the dermatological field, as it easily absorbable by the skin. Wound healing requires a complex interaction and coordination of different cells and molecules. Any alteration in these highly coordinated events can lead to either delayed or excessive healing. The goal of this review is to elucidate the cellular mechanisms involved, upon treatment with ALA-PDT, in chronic wounds, which are often associated with social isolation and high costs in terms of care.
... NO can promote apoptosis in some cells and inhibit apoptosis in others. The local cellular environment plays a dynamic role in determining the nature of these types of regulation [91,92]. Important components of the microenvironment are as follows: the redox state of cells, glutathione, the presence of other oxygen-and nitrogen-centered radicals. ...
Article
Apoptosis is the main feature of inflammatory-fibroproliferative disorders of the vessel wall. Studies in animal models have shown that smooth muscle cells (SMCs) cultured from endarterectomy specimens from the affected area proliferate more slowly and display higher apoptotic indices than SMCs derived from the normal vessel wall. Apoptotic cells were found in the destabilized atherosclerotic plaques, as well as in the samples with restenosis of the reconstruction area. Injury to the vessel wall causes two waves of apoptosis. The first wave is the rapid apoptosis in the media that occurs within a few hours after injury and leads to a marked reduction in the number of vascular wall cells. The second wave of apoptosis occurs much later (from several days to weeks) and is limited by the SMCs within the developing neointima. Up to 14% of the neointimal SMCs undergo apoptosis 20 days after balloon angioplasty. Ligation of the external carotid artery in a rabbit model led to a marked decrease in blood flow in the common carotid artery, which correlated with the increased apoptosis of endothelial cells and SMCs. Angioplasty-induced death of SMCs is regulated by a redox-sensitive signaling pathway, and topical administration of antioxidants can minimize vascular cell loss. On the whole, studies show that apoptosis is prevalent in vascular lesions, controlling the viability of both inflammatory and vascular cells, determining the cellular composition of the vessel wall. The main markers of apoptosis (Fas, Fas ligand, p53, Bcl-2, Bax) and cell proliferation (toll receptor) have been considered in the current review.
... In contrast, at higher concentrations, for example, during immune response, NO autoxidation becomes increasingly feasible kinetically. Thus, the third order kinetics behavior for NO autoxidation determines how this reactive molecule can play contrasting roles in oxygenated media, bioregulatory at low concentrations, but potentially cytotoxic when generated at higher concentrations in response to pathogen invasion [78,79]. ...
Article
In this article we discuss the fundamental chemical and physical properties of NO and related nitrogen oxides (NO2⁻, NO2, N2O3, etc.) under solution conditions relevant to mammalian biology.
... Excessive inflammation leads to the release of an enormous number of inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6), which cause cell necrosis and tissue damage, thus aggravate inflammation (Medzhitov, 2008;Chu et al., 2020). Nitric oxide (NO) is a small molecule compound that regulates neurotransmission and inflammatory response (Lee et al., 2017). In mammals, NO is produced by three different nitric oxide synthases (NOS), namely inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS). ...
Article
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BackgroundM1 macrophage plays an important role in inflammatory reaction. In this study, potential anti-inflammatory effect of Phyllolobium chinense Fisch flavonoids (PCFF) was assessed via Zebrafish acute inflammation model in vivo and LPS-induced pro-inflammatory M1 macrophage model in vitro.Methods The quality control of P. chinense Fisch flavonoids (PCFF) was analyzed by HPLC. Anti-inflammatory effect of PCFF on the acute injured zebrafish was evaluated by the migration of fluorescence labeled macrophages and neutrophils, and the gene expression of inflammatory factors. In addition, the anti-inflammatory mechanism of PCFF was investigated by the related gene expression and related signaling pathway regulation of pro-inflammatory mediators in LPS-induced pro-inflammatory M1 RAW264.7 macrophage.ResultsP. chinense Fisch flavonoids (PCFF) markedly suppressed macrophage and neutrophil migration and iNOS gene expression in acute injured zebrafish with tail-cutting. PCFF significantly inhibited NO overproduction and iNOS gene overexpression in LPS-sitimulated pro-inflammatory M1 RAW264.7 macrophages. What’s more, PCFF could evidently decrease p65 protein production, but had no effect on the production of P38, JNK and ERK1/2 proteins.ConclusionP. chinense Fisch flavonoids (PCFF) have a remarkable inhibitory effect on the inflammatory response in acute injured zebrafish and LPS-stimulated M1 RAW264.7 macrophage. The pharmacological mechanism may be related to the regulation of NO overproduction and the inhibition of NF-κB/iNOS signaling pathway.
... The wide clinical potential of NO-therapy is associated with the regulatory functions of NO in normal and pathological conditions [4,34]. According to our research and the other data, the effectiveness of NO-therapy is based on the bactericidal action of nitric oxide and peroxynitrite (ONOO-). ...
Article
Background It is known that exogenous nitric oxide (NO) has a bactericidal effect, activates proliferation of fibroblasts, enhances angiogenesis and collagen synthesis, accelerates maturation of granulation tissue. Objective The purpose of this research was to study the dose-dependent therapeutic effect of nitric oxide (NO) in NO-containing gas flows (NO-CGF) applied to the wound surfaces. Methods The experiment was performed in 36 white Wistar rats with full-thickness skin wounds with an area size of 3 cm². We used Plason device modified with an experimental plasma-chemical generator of nitric oxide (manipulator) for the following parameters of NO-CGF in the wound surface area: mass flow rate of nitric oxide 2.0 mg/s, axial gas flow velocity 9 m/s, nitric oxide content 1340 ppm, nitrogen dioxide content 108 ppm, gas flow temperature 39°C. Each group included 6 animals. The mass of nitric oxide delivered to the wound varied by the exposure time of 15, 30, 60, 120 and 360 seconds and was 30, 60, 120, 240 and 720 mg, respectively. Wounds were left untreated in the control group. Wound tissues were excised 4 days after the operation and studied with histological, semi-quantitative and morphometric methods. Results Histological analysis revealed beneficial effects of NO therapy on wound healing, including reduction of microcirculatory disorders and exudate volume and acceleration of fibroblast proliferation and collagen synthesis. We proved that the most effective stimulation of the wound healing process occurred with a duration of exposure of 120 and 360 seconds (integral mass doses of NO on the wound surfaces were 240 mg and 720 mg, relative mass doses of NO per wound areas were 80 mg/cm² and 240 mg/cm², respectively). We did not identify the overdose phenomena after prolonged exposure of the wound to NO. Conclusion Our results are important for further improvement of the technique and the standardization of NO-therapy for the treatment of wounds, inflammatory and destructive processes in clinical practice.
... It is interesting to note that, although the presence of MSCs tended to mitigate the polarization discrepancy of macrophages caused by different matrix cues (Fig. 5), the expression of Nos2 in macrophages cultured on nanofibrous matrix was still significantly lower than that in macrophages on smooth matrix. As a primary marker for M1 polarization, iNOS (inducible nitric oxide synthase) is an important enzyme in charge of producing nitric oxide (NO), which acts as an effector in inflammation in defense of microbial infection [45]. Besides the microbial killing function, NO as an important gaseous transmitter plays a complicated role in inflammation. ...
Article
An in-depth understanding of biomaterial cues to selectively polarize macrophages is beneficial in the design of “immuno-informed” biomaterials that positively interact with the immune system to dictate a favorable macrophage response following implantation. Given the promising future of ECM-mimicking nanofibrous biomaterials in biomedical application, it is essential to elucidate how their intrinsic cues, especially the nanofibrous architecture, affect macrophages. In the present study, we evaluated how the nanofibrous architecture of a gelatin matrix modulated macrophage responses from the perspectives of cellular behaviors and a transcriptome analysis. In our results, the nanofibrous surface attenuated M1 polarization and down-regulated the inflammatory responses of macrophages compared with a smooth surface. Besides, the cell-material interaction was up-regulated and the adhered macrophages tended to maintain an original, non-polarized state on the nanofibrous matrix. Accordingly, whole transcriptome analysis revealed that nanofibrous architecture up-regulated the pathways related to ECM-receptor interaction and down-regulated pathways related to pro-inflammation. This study provides a panoramic view of the interaction between macrophages and nanofibers, and offers valuable information for the design of immunomodulatory ECM-mimicking biomaterials for tissue regeneration.
... Bechara and associates [147] also demonstrated that monocyte-derived DCs, when stimulated with Ni, were able to release IL-23, with the polarization of the immune response toward Th17 cells [148]. Jacob and associates [149] documented that monocytes stimulated with Ni were able to produce TNF-α, ROS and NO [150][151][152][153][154]. ...
Article
Background Heavy metals [arsenic, aluminium, cadmium, chromium, cobalt, lead, nickel (Ni), palladium and titanium] are environmental contaminants able to impact with host human cells, thus, leading to severe damage. Objective In this review, the detrimental effects of several heavy metals on human organs will be discussed and special emphasis will be placed on Ni. In particular, Ni is able to interact with Toll-like receptor (TLR)-4 on immune and non immune cells, thus, triggering the cascade of pro-inflammatory cytokines. Then, inflammatory and allergic reactions mediated by Ni will be illustrated within different organs, even including the central nervous system, airways and the gastrointestinal system. Discussion Different therapeutic strategies have been adopted to mitigate Ni-induced inflammatory-allergic reactions. In this context the ability of polyphenols to counteract the inflammatory pathway induced by Ni on peripheral blood leukocytes from Ni-sensitized patients will be outlined. In particular, polyphenols are able to decrease serum levels of interleukin (IL)-17, while increasing levels of IL-10. These data suggest that the equilibrium between T regulatory cells and T helper 17 cells is recovered with IL-10 acting as an anti-inflammatory cytokine. In the same context, polyphenols reduced elevated serum levels of nitric oxide, thus, expressing their anti-oxidant potential. Finally, the carcinogenic potential of heavy metals, even including Ni, will be highlighted. Conclusion Heavy metals and, in particular, Ni are very spread in the environment. Nutritional approaches seem to represent a novel option in the treatment of Ni-induced damage and, among them, polyphenols should be taken into consideration for their anti-oxidant and anti-inflammatory activities.
... Nitric oxide (NO) is a molecular signal that regulates cardiovascular functions and has an inhibitory effect on leukocyte adhesion, platelet aggregation, and the proliferation of smooth muscle cells. When myocardial tissue is damaged by ischemia and hypoxia, iNOS expression is induced to synthesize NO and regulate myocardial damage (27). This shows that PRDX6 overexpression can improve the inflammatory response of myocardial tissue induced by doxorubicin, although the mechanism of action needs further study. ...
Article
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Background: Adriamycin is an anthracycline drug used to treat a variety of tumors. Adriamycin has a much stronger affinity for myocardial tissue than other body tissues. Cancer patients treated with adriamycin are prone to toxic damage to heart tissue. Peroxiredoxin 6 (PRDX6) is a novel antioxidant enzyme in metabolic diseases, the aim of this study was to investigate the role of PRDX6 in myocardial injury. Methods: Sixty male specific-pathogen-free Wistar rats were enrolled and divided equally into the control group (control), the Adriamycin group, the Adriamycin + empty vector lentivirus (Adriamycin + LV) group, and the Adriamycin + Peroxiredoxin 6 overexpression (Adriamycin + PRDX6) group. Western blot, reverse transcription-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, hematoxylin and eosin staining (HE) and immunohistochemistry were used in this research. Results: The myocardial tissues of the Adriamycin group had significantly lower expression levels of PRDX6 and PRDX6 mRNA than those of the control group, and the myocardial tissues of the Adriamycin + PRDX6 rats had significantly higher expression levels of PRDX6 and PRDX6 mRNA than those of the Adriamycin + LV group. Serum creatine kinase isoenzyme (CK-MB), myoglobin (Mb), cardiac troponin I (cTnI), myocardial injury, positive rate of caspase-3, B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) levels, malondialdehyde (MDA), lactate dehydrogenase (LDH), IL-1β, IL-6, inducible nitric oxide synthase (iNOS), and IL-4 proteins in the adriamycin-induced rats were significantly higher than those in the control group, while superoxide dismutase (SOD) activity was significantly lower than that in the control group. PRDX6 overexpression reversed the above results. Conclusions: PRDX6 overexpression can alleviate adriamycin-induced myocardial injury in rats, which may be related to oxidative stress regulation and the levels of inflammatory factors.
... The LPS-stimulated medium has also determined an increase in the oxidative stress, as demonstrated by the upregulation of the iNOS expression. NOS enzymes synthesize nitric oxide, which can lead to peroxynitrite formation, with consequent DNA damage [42]. It is known that LPS may lead to an increase in iNOS expression [43]. ...
Article
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Background and Objectives: Neuroinflammation is associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In this study, we investigate the anti-inflammatory, anti-oxidant, and anti-apoptotic properties of two non-psychoactive phytocannabinoids, cannabigerol (CBG) and cannabidiol (CBD). Materials and Methods: The motoneuron-like cell line NSC-34 differentiated by serum deprivation and with the additional treatment of all-trans retinoic acid (RA) is a valid model to investigate molecular events linked to neurodegeneration in ALS. Results: Pre-treatment with CBG (at 2.5 and 5 µM doses) alone and in combination with CBD (at 2.5 and 5 µM doses) was able to reduce neuroinflammation induced by a culture medium of LPS-stimulated macrophages. In particular, the pre-treatment with CBD at a 5 µM dose decreased TNF-α levels and increased IL10 and IL-37 expression. CBG–CBD association at a 5 µM dose also reduced NF-kB nuclear factor activation with low degradation of the inhibitor of kappaB alpha (IkBα). CBG and CBD co-administered at a 5 µM dose decreased iNOS expression and increased Nrf2 levels. Furthermore, the pre-treatment with the association of two non-psychoactive cannabinoids downregulated Bax protein expression and upregulated Bcl-2 expression. Our data show the anti-inflammatory, anti-oxidant, and anti-apoptotic effects PPARγ-mediated. Conclusions: Our results provide preliminary support on the potential therapeutic application of a CBG–CBD combination for further preclinical studies.
... NO regulates vasodilation and is an important part of the inflammatory reaction [21]. Leachate from wheat malt produced no significant effect on lipopolysaccharide-induced NO formation, but all the individual phenols except vanillic acid gave a significant effect (Table 1). ...
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Extensively malted cereals counteract enterotoxic diarrhea and inflammatory bowel diseases. This effect depends on a protein called antisecretory factor (AF), which is secreted into the blood as a larger complex known as the compleasome. In this study, we identified anti-inflammatory substances in malt and assayed their capacity to induce AF. Guaiacol and quercetin inhibited inflammation in a mouse footpad model, while catechin, sinapic acid, ferulic acid, and quercetin inhibited nitric oxide formation in RAW 264.7 cells. The proteasome activity in these cells was inhibited by vanillic acid and quercetin but not by the other tested phenols. As the transient receptor potential vanilloid 1 (TRPV1) might be involved in AF induction, the TRPV1 antagonist capsazepine was tested and shown to inhibit inflammation in mouse paw and nitric oxide formation. Catechin, ferulic acid, and sinapic acid induced AF in rat blood, and these substances were all increased in malt compared to control wheat. These phenols might therefore be of particular importance for the beneficial effect of malted cereals on inflammatory diseases. Our results further suggest that TRPV1 might play a role in the anti-inflammatory activity of phenols via the induction of AF.
... Bl_NOS transcription also reduced with HDACi treatment. NOS is essential for the generation of nitric oxide, which has important roles in pluripotency and immune responses (Beltran-Povea et al., 2015;Cencioni et al., 2018;Lee et al., 2017). Bl_NOS mRNA is expressed in multiple cell types including haemoblasts and the regeneration niche. ...
Article
The colonial tunicate Botrylloides leachii is exceptional at regenerating from a piece of vascular tunic after loss of all adults from the colony. Previous transcriptome analyses indicate a brief period of healing before regeneration of a new adult (zooid) in as little as 8-10 days. However, there is little understanding of how the resulting changes to gene expression, required to drive regeneration, are initiated and how the overall process is regulated. Rapid changes to transcription often occur in response to chromatin changes, mediated by histone modifications such as histone acetylation. Here, we investigated a group of key epigenetic modifiers, histone deacetylases (HDAC) that are known to play an important role in many biological processes such as development, healing and regeneration. Through our transcriptome data, we identified and quantified the expression levels of HDAC and histone acetyltransferase (HAT) enzymes during whole body regeneration (WBR). To determine if HDAC activity is required for WBR, we inhibited its action using valproic acid (VPA) and Trichostatin A (TSA). HDAC inhibition prevented the final morphological changes normally associated with WBR and resulted in aberrant gene expression. B. leachii genes including Slit2, TGF-β, Piwi and Fzd4 all showed altered mRNA levels upon HDAC inhibition in comparison to the control samples. Additionally, atypical expression of Bl_Piwi was found in immunocytes upon HDAC inhibition. Together, these results show that HDAC function, specifically HDAC I/IIa class enzymes, are vital for B. leachii to undergo WBR successfully.
... In this context, our group has been investigating the role of neutrophil-derived NO to sepsis outcome. Neutrophils are essential for eradicating a bacterial infection and avoiding bacterial spread [12]. Consistent with this concept, a reduction on the number of neutrophils was observed in the focus of infection during experimental severe sepsis induced by cecum ligation and the puncture model, as well as by intraperitoneal administration of Grampositive, Gram-negative or cecal bacteria [13][14][15]. ...
Article
Nitric oxide (NO) is produced by enzymatic activity of neuronal (nNOS), endothelial (eNOS), and inducible nitric oxide synthase (iNOS) and modulates a broad spectrum of physiological and pathophysiological conditions. The iNOS isoform is positively regulated at transcriptional level and produces high levels of NO in response to inflammatory mediators and/or to pattern recognition receptor signaling, such as Toll-like receptors. In this review, we compiled the main contributions of our group for understanding of the role of NO in sepsis and arthritis outcome and the peripheral contributions of NO to inflammatory pain development. Although neutrophil iNOS-derived NO is necessary for bacterial killing, systemic production of high levels of NO impairs neutrophil migration to infections through inhibiting neutrophil adhesion on microcirculation and their locomotion. Moreover, neutrophil-derived NO contributes to multiple organ dysfunction in sepsis. In arthritis, NO is chief for bacterial clearance in staphylococcal-induced arthritis; however, it contributes to articular damage and bone mass degradation. NO produced in inflammatory sites also downmodulates pain. The mechanism involved in analgesic effect and inhibition of neutrophil migration is dependent on the activation of the classical sGC/cGMP/PKG pathway. Despite the increasing number of studies performed after the identification of NO as an endothelium-derived relaxing factor, the underlying mechanisms of NO in inflammatory diseases remain unclear.
... While production of nitric oxide (NO) and mitochondrial reactive oxygen species (mROS) are often central in the clearance of pathogenic infections prior to development of adaptive immunity, each are known to have deleterious effects on the magnitude of activation as well as the phenotype and survival of innate and adaptive immune cells [5][6][7][8][9][10]. For example, mice infected with lymphocytic choriomeningitis virus (LCMV) which develop severe lymphopenia have a poor adaptive immune response (e.g., no neutralizing antibody) that has been attributed to the large amounts of NO produced by inflammatory monocytes in the lymph nodes [11]. ...
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Background One of the most concerning public health issues, related to vaccination and disease prevention, is the inability to induce durable immune responses following a single-dose immunization. In this regard, the nature of the inflammatory environment induced by vaccine adjuvants can negatively impact the resulting immune response. To address these concerns, new strategies to vaccine design are needed in order to improve the outcomes of immune responses, particularly in immunologically disadvantaged populations. Methods Comparisons of the scope of innate immune activation induced by TLR agonists versus cyclic dinucleotides (CDNs) was performed. Their effects on the activation characteristics (e.g., metabolism, cytokine secretion) of bone marrow derived dendritic cells (BMDCs) were studied. In addition, the differential effects on in vivo induction of antibody responses were measured. Results As compared to TLR ligands, the stimulation of BMDCs with CDNs induced distinctly different metabolic outcomes. Marked differences were observed in the production of nitric oxide (NO) and the cytokine BAFF. These distinct differences were correlated with improved (i.e., more rapid and persistent) vaccine antibody responses in both aged and young mice. Conclusions Our results illustrate that the innate immune pathway targeted by adjuvants can critically impact the outcome of the immune response post-vaccination. Specifically, CDN stimulation of APCs induced an activation phenotype that was characterized by decreased innate effector molecule production (e.g., NO) and increased BAFF. This was attributed to the induction of an innate inflammatory environment that enabled the host to make the most of the existing B lymphocyte potential. The use of adjuvants that differentially engage mechanisms of innate immune activation would be particularly advantageous for the generation of robust, single dose vaccines. The results of this study demonstrated that CDNs induced differential innate activation and enhanced vaccine induced antibody responses in both young and aged mice.
... The expressions of Tnf and Ccr7 showed a similar but nonsignificant tendency. Nos2 is the coding gene of inducible nitric oxide synthase (iNOS), which can produce high levels of NO for pathogen clearance [41]. Il1b and Tnf encode IL-1β and TNF-α, respectively, which are two important proinflammatory cytokines and are crucial treatment targets for certain inflammation-related conditions, such as autoimmune diseases [42,43]. ...
Article
Extracellular matrices (ECMs) derived from native tissues/organs have been used as biomaterials for tissue engineering and regenerative medicine in a wide range of preclinical and clinical settings. The success or failure of these applications is largely contingent on the host responses to the matrices in vivo. Despite retaining their native structural and functional proteins, bone ECM-based transplants have been reported to evoke adverse immune responses in many cases; thus, optimizing the immunomodulatory properties of bone ECMs is critical for ensuring downstream regenerative outcomes. Using a simple digestion-neutralization protocol, we transformed the commonly used bone-derived filler particles into gel bioscaffolds. Instead of inducing macrophages toward proinflammatory (M1) polarization, as reported in the literature and confirmed in the present study for ECM particles, the ECM gels were found to be more likely to polarize macrophages toward regulatory/anti-inflammatory (M2) phenotypes, leading to enhanced tissue regeneration in a rat periodontal defect model. The present work demonstrates a simple, practical and economical strategy to modify the immunomodulatory properties of bone ECMs before their in vivo transplantation and hence has important implications that may facilitate the use of ECM-based bioscaffolds derived from diverse sources of tissues for regenerative purposes.
... This result is similar to others in the literature [10,19]. The augmented iNOS expression observed in our experimental conditions suggests an increase in inflammatory processes due to liver injury since iNOS is mainly induced by the presence of endotoxin and proinflammatory cytokines [20]. Also, NO plays an important negative feedback regulatory role on endothelial NO synthase and, therefore, vascular endothelial cell function, which explains the lower local production of NO [21]. ...
Article
Liver cirrhosis is associated with a wide range of cardiovascular abnormalities including hyperdynamic circulation and cirrhotic cardiomyopathy. The pathogenic mechanisms of these cardiovascular changes are multifactorial and include vascular dysregulations. Aim: The present study tested the hypothesis that the systemic vascular hyporesponsiveness in thioacetamide (TAA)-induced liver injury model is dependent on nitric oxide (NO) and cyclooxygenase (COX) derivatives. Main methods: Wistar rats were treated with TAA for eight weeks to induce liver injury. Key findings: The maximal contractile response in concentration-effect curves to phenylephrine was decreased in aorta from TAA-treated rats, but no differences were found in aorta without endothelium, suggesting an endothelium-dependent mechanism in decreased contractile response. There was no difference in the contractile response with and without L-NAME (N(ω)-nitro-l-arginine methyl ester) in rats with liver injury, showing that the TAA treatment impairs NO synthesis. Pre-incubation of the aorta with indomethacin, a COX-inhibitor, normalized the reduced contractile response to phenylephrine in arteries from TAA group. Also, COX-2 and iNOS (inducible nitric oxide syntase) protein expression was increased in aorta from TAA group compared to control group. Animals submitted to TAA treatment had a reduction in systolic blood pressure. Our findings demonstrated that liver injury induced by TAA caused a decrease in aortic contractile response by a COX-dependent mechanism but not by NO release. Also, it was demonstrated an inflammatory process in the aorta of TAA-treated rats by increased expression of COX-2 and iNOS. Significance: Therefore, there is an essential contribution of COX-2 activation in extra-hepatic vascular dysfunction and inflammation present in cirrhosis induced by TAA.
... Although it is difficult to find specific phenotypical markers to delineate M0, M1, and M2 macrophages, recent findings in mouse provide evidence that some surface markers can be considered as representative of each subtype of macrophage, such as CD38 for M1 and early growth response protein 2 for M2 (58). Taken together, the cytokine pathways, nitric oxide (NO) and polyamine levels, may explain why there is more than a simple duality of microbicidal/pro-inflammatory properties versus cell growth/ anti-inflammatory properties in the macrophage subpopulations (59,60). Actually, M1 and M2 phenotypes often coexist, and other terms have emerged to identify non-classical activation phenotypes such as M2a or M2b, the latter representing alternative activated macrophages that express small amounts of arginase 1 (56). ...
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Mononuclear phagocytes (monocytes, dendritic cells, and macrophages) are among the first host cells to face intra- and extracellular protozoan parasites such as trypanosomatids, and significant expansion of macrophages has been observed in infected hosts. They play essential roles in the outcome of infections caused by trypanosomatids, as they can not only exert a powerful antimicrobial activity but also promote parasite proliferation. These varied functions, linked to their phenotypic and metabolic plasticity, are exerted via distinct activation states, in which l-arginine metabolism plays a pivotal role. Depending on the environmental factors and immune response elements, l-arginine metabolites contribute to parasite elimination, mainly through nitric oxide (NO) synthesis, or to parasite proliferation, through l-ornithine and polyamine production. To survive and adapt to their hosts, parasites such as trypanosomatids developed mechanisms of interaction to modulate macrophage activation in their favor, by manipulating several cellular metabolic pathways. Recent reports emphasize that some excreted–secreted (ES) molecules from parasites and sugar-binding host receptors play a major role in this dialog, particularly in the modulation of the macrophage’s inducible l-arginine metabolism. Preventing l-arginine dysregulation by drugs or by immunization against trypanosomatid ES molecules or by blocking partner host molecules may control early infection and is a promising way to tackle neglected diseases including Chagas disease, leishmaniases, and African trypanosomiases. The present review summarizes recent knowledge on trypanosomatids and their ES factors with regard to their influence on macrophage activation pathways, mainly the NO synthase/arginase balance. The review ends with prospects for the use of biological knowledge to develop new strategies of interference in the infectious processes used by trypanosomatids, in particular for the development of vaccines or immunotherapeutic approaches.
... 45,46 One component of the overt inflammatory responses induced by pathogens or TLR-based adjuvants is the production of microbicidal innate immune effector molecules such as NO. 47,48 Although NO is important for the innate immune response to overcome acute pathogenic infections, 49 it has negative autocrine or paracrine effects on APCs. When NO is produced by BMDCs, it inhibits the electron transport chain (ie, oxidative phosphorylation) leading to a dependence on aerobic glycolysis for ATP production and cellular survival. ...
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Introduction: Adjuvants and immunotherapies designed to activate adaptive immunity to eliminate infectious disease and tumors have become an area of interest aimed at providing a safe and effective strategy to prevent or eliminate disease. Existing approaches would benefit from the development of immunization regimens capable of inducing efficacious cell-mediated immunity directed toward CD8+ T cell-specific antigens. This goal is critically dependent upon appropriate activation of antigen-presenting cells (APCs) most notably dendritic cells (DCs). In this regard, polyanhydride particles have been shown to be effectively internalized by APCs and induce activation. Methods: Here, a prophylactic vaccine regimen designed as a single-dose polyanhydride nanovaccine encapsulating antigen is evaluated for the induction of CD8+ T cell memory in a model system where antigen-specific protection is restricted to CD8+ T cells. Bone marrow-derived dendritic cells (BMDCs) are used as an in vitro model system to evaluate the magnitude and phenotype of APC activation. Primary DCs, particularly those with described ability to activate CD8+ T cells, are also evaluated for their in vitro responses to polyanhydride nanoparticles. Results: Herein, polyanhydride nanoparticles are shown to induce potent in vitro upregulation of costimulatory molecules on the cell surface of BMDCs. In contrast to the classically used TLR agonists, nanoparticles did not induce large amounts of pro-inflammatory cytokines, did not induce characteristic metabolic response of DCs, nor produce innate antimicrobial effector molecules, such as nitric oxide (NO). The polyanhydride nanovaccine results in protective CD8+ T cell responses as measured by inhibition of tumor progression and survival. Discussion: Together, these results suggest that the use of a polyanhydride-based nanovaccine can be an effective approach to inducing antigen-specific CD8+ T cell memory by providing antigen delivery and DC activation while avoiding overt inflammatory responses typically associated with traditional adjuvants.
... Ensuite, le NO est impliqué dans un certain nombre de maladies inflammatoires et autoimmunes (Lee et al., 2017). Par exemple, l'induction de la iNOS par les LPS et les cytokines proinflammatoires contribue aux maladies inflammatoires de l'intestin (Kolios et al., 2004;Tun et al., 2014). ...
Thesis
Les Escherichia coli entérohémorragiques (EHEC) représentent un enjeu majeur en santé publique. En effet, ces pathogènes sont responsables chaque année de milliers de cas de toxi-infections alimentaires à travers le monde et peuvent engendrer des complications graves, notamment des atteintes rénales chez les jeunes enfants et cérébrales chez les personnes âgées. Actuellement, le principal problème réside dans le fait que les traitements thérapeutiques disponibles sont limités puisque l’antibiothérapie peut favoriser le développement des complications liées à l’infection. Il est donc primordial et d’actualité de mettre en évidence les facteurs bactériens associés à la virulence des EHEC et de comprendre les interactions entre le pathogène et l’hôte afin de développer des stratégies thérapeutiques visant à éliminer le pathogène et limiter l’apparition des symptômes graves. Ainsi, le premier objectif de cette thèse était d’identifier de nouveaux facteurs bactériens potentiellement impliqués dans le processus infectieux. L’utilisation de la technologie RIVET sur la souche de référence O157:H7 EDL933 en modèle murin, a permis de mettre en évidence 31 gènes dont l’expression est spécifiquement induite lors de l’infection. La caractérisation de ces gènes a démontré que certains codent des facteurs de niche qui pourraient accroître le potentiel des souches d’EHEC à s’adapter à l’environnement intestinal et ainsi participer à la virulence du pathogène. Le second volet de cette thèse avait pour but de caractériser in vivo la réponse des EHEC au monoxyde d’azote (NO), un médiateur de la réponse immunitaire de l’hôte, et ainsi d’évaluer le potentiel rôle protecteur du NO lors d’une infection en modèle murin. En utilisant une souche d’EHEC rapportant la présence de NO, nous avons démontré que le NO est produit dès les premiers stades de l’infection et que celui-ci limite l’adhésion du pathogène à la muqueuse colique. En revanche, nous avons également mis en évidence un effet néfaste du NO pour l’hôte puisqu’il favorise la production des Shigatoxines (Stx), le facteur de virulence majeur des EHEC, conduisant au développement d’un dysfonctionnent rénal. Enfin, nous avons montré l’importance de la NO réductase NorVW dans la virulence de certaines souches d’EHEC. En effet, l’inactivation de l’opéron norVW chez la souche O157:H7 620 réduit la capacité du pathogène à coloniser efficacement le tractus digestif et à produire Stx. Cette observation est toutefois souche dépendante et suggère que la réponse des EHEC au stress nitrosant lors d’une infection est complexe et probablement multifactoriel. L’ensemble de ces travaux contribue à une meilleure compréhension du processus infectieux des EHEC, une étape indispensable au développement de futures stratégies anti-infectieuses.
... To assess the possibility of modulating hepatic infection by Plasmodium through dietary supplementation, we sought to increase the bioavailabilty of Arg (R) as a physiological substrate for the synthesis of nitric oxide (NO), which is a key mediator of immune responses (Lee et al., 2017;Roth, 1992;Bogdan, 2001). To achieve this, we supplemented the drinking water of C57BL/6J mice with 2.5% (w/v) of Arg and the arginase inhibitors Lys (K) and Val (V), either individually or in combinations of equal concentrations (RV, KV, RK, and RKV). ...
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Plasmodium parasites, causative agents of malaria, scavenge host nutrients to sustain their intracellular replication. Modulation of the host's nutritional status can potentially help control infection by limiting the parasite's access to nutrients, or by boosting the immune system. Here, we show that dietary supplementation of mice employing a combination of arginine (R) with two additional amino acids, lysine (K) and valine (V), termed RKV, significantly decreases Plasmodium liver infection. RKV supplementation results in the elimination of parasites at a late stage of their development in the liver. Our data employing genetic knockout mouse models and in vivo depletion of specific cell populations suggest that RKV supplementation boosts the host's overall innate immune response, and that parasite elimination is dependent on MyD88 signaling in immune cells. The immunostimulatory effect of RKV supplementation opens a potential role for dietary supplementation as an adjuvant for prophylaxis or immunization strategies against Plasmodium infection.
... tems of the body, leading to tissue damage, the development of inflammation, and inducing cell death [26]. Hence, to control its production is a principal role in an anti-inflammatory investigation. ...
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This study aimed at preparing and characterizing thymol, eugenol, and piperine-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles and evaluating the effect on inflammatory mediators secretion and apoptosis in Raw 264.7 macrophage cells. Nanoparticles were produced by the solvent evaporation technique. Dynamic light scattering and scanning electron microscopy were used to study the physicochemical characteristics. Raw 264.7 macrophage cells were used as a model for in vitro assays. The 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium assay was used to determine the cytotoxicity of the formulated nanoparticles. An annexin V apoptosis detection kit was used to assess apoptosis. Nitric oxide production was determined using the Griess reagent, and the inflammatory mediators level was evaluated with Th1/Th2 cytokine and fluorometric cyclooxygenase kits. The loaded nanoparticles showed a particle size around 190 nm with a low polydispersity between 0.069 and 0.104 and a zeta potential between–1.2 and–9.5 mV. Reduced cytotoxicity of nanoparticles compared to free molecules against Raw 264.7 macrophage cells was observed and seemed to occur through a mechanism associated with apoptosis. A decrease in cyclooxygenase enzyme activity with an increasing concentration was observed. Both free molecules and nanoparticles showed their capacity to modulate the inflammatory process mostly by inhibiting the investigated inflammatory cytokines. The data presented in this study indicate that thymol and piperine-loaded poly(D,L-lactic-co-glycolic acid nanoparticles could serve as a novel anti-inflammatory colloidal drug delivery system with reduced toxicity. However, further study should be considered to optimize the formulation’s loading capacity and thereby probably enhance their bioactivity in treating inflammatory diseases.
... However, this effect was not seen in EC-specific Arg1 knockout mice (Fouda et al., 2018). It is postulated that the neuroprotective effects of Arg1 are due to the reduced availability of L-arginine for iNOS, which functions to amplify the pro-inflammatory state through increased macrophage polarization to the M1-like, proinflammatory phenotype (Lee et al., 2017). Elevated Arg1 reduces L-arginine levels, the substrate necessary for iNOS to maintain high NO production and inflammation. ...
Article
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Diabetic retinopathy (DR) and diabetic macular edema (DME) are retinal complications of diabetes that can lead to loss of vision and impaired quality of life. The current gold standard therapies for treatment of DR and DME focus on advanced disease, are invasive, expensive, and can trigger adverse side-effects, necessitating the development of more effective, affordable, and accessible therapies that can target early stage disease. The pathogenesis and pathophysiology of DR is complex and multifactorial, involving the interplay between the effects of hyperglycemia, hyperlipidemia, hypoxia, and production of reactive oxygen species (ROS) in the promotion of neurovascular dysfunction and immune cell polarization to a proinflammatory state. The pathophysiology of DR provides several therapeutic targets that have the potential to attenuate disease progression. Current novel DR and DME therapies under investigation include erythropoietin-derived peptides, inducers of antioxidant gene expression, activators of nitric oxide/cyclic GMP signaling pathways, and manipulation of arginase activity. This review aims to aid understanding of DR and DME pathophysiology and explore novel therapies that capitalize on our knowledge of these diabetic retinal complications.
... This series of events indicates that anti-inflammatory activity in macrophages is important to prevent atherosclerosis. In macrophages, nitric oxide (NO) is produced by inducible nitric oxide synthase and cytokine stimulation [7]. NO functions in several physiological and pathological processes associated with acute and chronic inflammation [8,9]. ...
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Atherosclerosis is considered the major cause of cardiovascular and cerebrovascular diseases, which are the leading causes of death worldwide. Excessive nitric oxide production and inflammation result in dysfunctional vascular endothelial cells, which are critically involved in the initiation and progression of atherosclerosis. The present study aimed to identify a bioactive compound from Jerusalem artichoke leaves with anti-inflammatory activity that might prevent atherosclerosis. We isolated bioactive heliangin that inhibited NO production in LPS-induced macrophage-like RAW 264.7 cells. Heliangin suppressed ICAM-1, VCAM-1, E-selectin, and MCP expression, as well as NF-kB and IkBa phosphorylation, in vascular endothelial cells stimulated with TNF-a. These results suggested that heliangin suppresses inflammation by inhibiting excessive NO production in macrophages and the expression of the factors leading to the development of atherosclerosis via the NF-kB signaling pathway in vascular endothelial cells. Therefore, heliangin in Jerusalem artichoke leaves could function in the prevention of atherosclerosis that is associated with heart attacks and strokes.
... More recently, studies relate the chemo-and immunoresistance [7] in cancer cells with NO as a mediator for the events in the tumor microenvironment (TME) and as a bonafide molecular target [15,16]. This review summarizes the classical physiology of macrophages and polarization, iNOS activities, and presents evidence towards tumor-associated macrophage reprogramming and the various immunometabolic and therapeutic options using iNOS or NO-dependent strategies. ...
Article
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Nitric oxide and its production by iNOS is an established mechanism critical to tumor promotion or suppression. Macrophages have important roles in immunity, development, and progression of cancer and have a controversial role in pro-and antitumoral effects. The tumor micro-environment consists of tumor-associated macrophages (TAM), among other cell types that influence the fate of the growing tumor. Depending on the microenvironment and various cues, macro-phages polarize into a continuum represented by the M1-like pro-inflammatory phenotype or the anti-inflammatory M2-like phenotype; these two are predominant, while there are subsets and in-termediates. Manipulating their plasticity through programming or reprogramming of M2-like to M1-like phenotypes presents the opportunity to maximize tumoricidal defenses. The dual role of iNOS-derived NO also influences TAM activity by repolarization to tumoricidal M1-type phenotype. Regulatory pathways and immunomodulation achieve this through miRNA that may inhibit the immunosuppressive tumor microenvironment. This review summarizes the classical physiology of macrophages and polarization, iNOS activities, and evidence towards TAM reprogramming with current information in glioblastoma and melanoma models, and the immunomodulatory and therapeutic options using iNOS or NO-dependent strategies.
... Three isoforms of NOS have been identified; two of them (endothelial NOS [10,11] and neuronal NOS [12,13]) are expressed constitutively, while the last one is inducible and is mainly involved in the inflammatory/immune response [14][15][16][17]. ...
Article
L-Arginine is involved in many different biological processes and recent reports indicate that it could also play a crucial role in the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, we present an updated over-view of the current evidence on the functional contribution of L-Arginine in COVID-19, describing its actions on endothelial cells and the immune system and discussing its potential as a therapeu-tic tool emerged from recent clinical experimentations.
... The results ( Figure 8D-F) suggested both ISM and MER remarkably inhibited TNF-α, IL-6, and IL-1β mRNA level in a dose-dependent manner at concentrations of 200 µM and 100 µM, while MH significantly decreased TNF-α transcription only at higher concentrations up to 200 µM, indicating that MH has a weaker inhibitory effect on inflammatory cytokines transcription. However, iNOS expression is also induced in response to other inflammatory stimuli such as cytokines [22]. Thus, although MH can significantly suppress LPS-induced iNOS mRNA transcription, the release of cytokines such as IL-6 and IL-1β could also stimulate iNOS overexpression and lead to the increase in NO produce. ...
Article
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C-prenyl coumarins (C-PYCs) are compounds with similar structures and various bioactivities, which are widely distributed in medicinal plants. Until now, the metabolic characterizations of C-PYCs and the relationship between metabolism and bioactivities remain unclear. In this study, ultra-performance chromatography electrospray ionization quadrupole time-of-flight mass spectrometry-based metabolomics (UPLC-ESI-QTOF-MS) was firstly used to determine the metabolic characterizations of three C-PYCs, including meranzin hydrate (MH), isomeranzin (ISM), and meranzin (MER). In total, 52 metabolites were identified, and all of them were found to be novel metabolites. Among these metabolites, 10 were from MH, 22 were from ISM, and 20 were from MER. The major metabolic pathways of these C-PYCs were hydroxylation, dehydrogenation, demethylation, and conjugation with cysteine, N-acetylcysteine, and glucuronide. The metabolic rate of MH was much lower than ISM and MER, which was only 27.1% in MLM and 8.7% in HLM, respectively. Additionally, recombinant cytochrome P450 (CYP) screening showed that CYP1A1, 2B6, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of metabolites. Further bioactivity assays indicated that all of these three C-PYCs exhibited anti-inflammatory activity, but the effects of ISM and MER were slightly higher than MH, accompanied by a significant decrease in inflammatory cytokines transcription induced by lipopolysaccharide (LPS) in macrophages RAW 264.7. Taken together, the metabolic characterizations of the three C-PYCs suggested that the side chain of the prenyl group may impact the metabolism and biological activity of C-PYCs.
... INOS is generally not expressed in cells. In macrophages, it is usually stimulated by some pathogens or cytokines to induce iNOS synthesis and produce nitric oxide (NO) [11], which can destroy cell stability, play a cytotoxic role, promote apoptosis, and also cause damage to surrounding tissues [12,13]. ...
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Background: Pseudomonas aeruginosa (PA) is one of the most common gram-negative opportunistic pathogens in nosocomial infection. The susceptible population is mainly patients with low immunity, such as pulmonary cystic fibrosis, chronic obstructive pulmonary disease, tumor, bronchiectasis and burn.Macrophages from monocytes are the main innate immune cells recruited to the site of inflammation. After infection leads to local inflammation, these macrophages recruited to the inflammatory site play a key role in the occurrence, diffusion and regression of inflammation. The proliferation ability of macrophages is reduced, which weakens the ability of macrophages to phagocytize pathogens, resulting in persistent infection and lung tissue injury.KLF6 plays an important role in the regulation of gene expression in inflammatory response, and it has been reported that there is a functional relationship between KLF6 and inducible nitric oxide synthase (iNOS). Our previous studies found that PA can induce the expression of KLF6 in lung tissue cells at the animal level, and may mediate apoptosis of lung tissue cells by regulating iNOS. In this study, the effect of PA supernatant on RAW264.7 cells apoptosis and the expression of KLF6 and iNOS were observed. In order to further study the role of iNOS in the apoptosis of RAW264.7 cells induced by PA supernatant, S-methylisothiourea sulfate (SMT) was added to the infected RAW264.7 cells to detect the expression level of iNOS, the change of NO content and the apoptosis of RAW264.7 cells. Methods: The MTT assay was used to detect the cell proliferation rate. Considering the different volume concentrations(the volume ratio of PA supernatant to complete medium) of PA supernatant, the experiment groups were divided into control group(add same volume of complete medium), 15% PA group, 30% PA group, 45% PA group, the cells in each group were treated for 12h and 24h respectively. In addition, SMT(+) group(SMT was the iNOS blocker) and SMT(-) group(without SMT) were designed for the experiment, the concentration was selected as 6μM according to the manual and preliminary results. According to MTT results, 30% PA supernatant treating for 24h was screened as the processing condition.Flow cytometry was used to detect the apoptosis rate. Hoechst 33342 staining was used to observe the nuclear morphological changes. Western blot was used to detect the expression of KLF6 and iNOS protein. The expression levels of KLF6 mRNA and iNOS mRNA were detected by real-time PCR. The NO content detection kit was used to detect the content of NO. Finally, the data analysis was conducted by using SPSS16.0 software. Results: When the PA supernatant at different concentrations treated the cells for the same time interval, the proliferation rate increased in a concentration-dependant manner, indicating that the PA supernatant inhibited the proliferation of RAW264.7 cells in a time-concentration-dependent manner. Meanwhile,the same results were achieved in flow cytometry and staining with Hoechst 33342 detected the same conclusion. Furthermore, the expression of KLF6 and iNOS protein, KLF6 mRNA and iNOS mRNA increased significantly after treatment, and NO content was statistically determined after treatment in a concentration-dependent manner and a time-dependent manner. Finally, compared with the SMT(-) group, the apoptosis rate was significantly decreased, and the NO content decreased after treatment with PA supernatant,. Conclusions: Our findings demonstrste tthat PA supernatant can inhibit the proliferation of RAW264.7 cells, and its inhibitory effect is concentration-time dependent. The mechanism by which PA induces apoptosis of RAW264.7 cells may be caused by the production of excess NO by KLF6 and iNOS. The reduction of iNOS expression has a protective effect on the cytotoxicity caused by PA supernatant infectied RAW264.7 cells.
... CAT could remove H 2 O 2 and improve the survival rate of cells when H 2 O 2 concentration was too high (Mahaseth & Kuzminov, 2017). Many immune cells in the body could express iNOS after being stimulated, then increase the secretion of NO, to dilate blood vessels and reduce the production of free radicals in the body (Lee et al., 2017). ...
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This study aimed to explore the protective effect of sulfated Codonopsis polysaccharides (SCP) on acute oxidative stress. SCP was modified by chlorosulfonic acid-pyridine method from Codonopsis polysaccharides (CP), which had 34.48% of sulfate content determined by ultrasonic-acidic barium chromate spectrophotometry. The analysis of Fourier transform-infrared spectroscopy (FT-IR) appeared an absorption peak of SCP at 811.91 cm⁻¹, which related to C-O-SO3. In vitro test, the antioxidant activities of CP and SCP was induced by H2O2 in RAW264.7 cells, results indicated that SCP and CP could significantly enhance the activity of superoxide dismutase (SOD), glutathione peroxidase (GDH-Px) and catalase (CAT), and nitric oxide (NO) and decrease the level of malondialdehyde (MDA), reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) secreted by RAW264.7 cells compared with modeling group (p < .05). The flow cytometry results also revealed that SCP and CP could markedly inhibit the apoptosis of macrophage induced by acute oxidative stress. In vivo test, 50% ethanol was used to induce mice acute oxidative stress, results indicated that the blood biochemical parameters in mice were restored to normal levels following administration of SCP and CP, andalanineamino transferase (ALT), aspartate transaminase (AST), total protein (TP), albumin (ALB), glucose (GLU), and creatinine (UREA) had significant differences compared with modeling group (p < .05). Quantitative real-time PCR analysis revealed that SCP and CP could promote the expression of Keap1 and Nrf2. In summary, both SCP and CP had protective effects against acute oxidative stress. Practical applications Oxidative stress is a kind of stress injury, which can cause a variety of diseases and accelerate physical aging. Codonopsis has many active components, among which Codonopsis polysaccharide has antioxidant effect. Recent studies have found that Codonopsis polysaccharides could be modified by sulfate molecules to obtain higher antioxidant activity. The modified Codonopsis polysaccharides could significantly promote the production of antioxidant enzymes (SOD, CAT, GDH-Px) and reduce the content of oxidative stress marks (ROS, MDA). Moreover, its antioxidant mechanism may be related to the Keap1 /Nrf2 signaling pathway. Therefore, SCP was an effective antioxidant, and could be used as a potential health food with antioxidant and anti-aging effects.
... The macrophages show positive cell surface expression of CD80, CD86, CD206, and show increased expression of suppressor of cytokine signaling-3 (SOCS3) and inducible nitric oxide synthase (iNOS) based on their phenotype [12][13][14][15]. iNOS, which produces nitric oxide (NO) is a key enzyme for the inflammatory role of macrophages, which predominantly participate in the pathogenesis of inflammatory diseases [12,16]. In periodontitis, an increased proportion of the M1 macrophages have been demonstrated implicating a destructive role played by these cells in periodontal pathogenesis [17]. ...
Article
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Background: Periodontitis is an inflammatory condition of the tooth-supporting structures initiated and perpetuated by pathogenic bacteria present in the dental plaque biofilm. In periodontitis, immune cells infiltrate the periodontium to prevent bacterial insult. Macrophages derived from monocytes play an important role in antigen presentation to lymphocytes. However, they are also implicated in causing periodontal destruction and bystander damage to the host tissues. Objectives: The objective of the present study was to quantify the cytokine profile of gingival crevicular fluid (GCF) samples obtained from patients with periodontitis. The study further aimed to assess if GCF of periodontitis patients could convert CD14+ monocytes into macrophages of destructive phenotype in an in vitro setting. The secondary objectives of the study were to assess if macrophages that resulted from GCF treatment of monocytes could affect the synthetic properties, stemness, expression of extracellular matrix proteins, adhesion molecules expressed by gingival stem cells, gingival mesenchymal stromal cells, and osteoblasts. Methods: GCF, blood, and gingival tissue samples were obtained from periodontitis subjects and healthy individuals based on specific protocols. Cytokine profiles of the GCF samples were analyzed. CD14+ monocytes were isolated from whole blood, cultured, and treated with the GCF of periodontitis patients to observe if they differentiated into macrophages. Further, the macrophages were assessed for a phenotype by surface marker analysis and cytokine assays. These macrophages were co-cultured with gingival stem cells, epithelial, stromal cells, and osteoblasts to assess the effects of the macrophages on the synthetic activity of the cells. Results: The GCF samples of periodontitis patients had significantly higher levels of IFN gamma, M-CSF, and GM-CSF. Administration of the GCF samples to CD14+ monocytes resulted in their conversion to macrophages that tested positive for CD80, CD86, and CD206. These macrophages produced increased levels of IL-1β, TNF-α, and IL-6. Co-culture of the macrophages with gingival stem cells, epithelial cells, and stromal cells resulted in increased cytotoxicity and apoptotic rates to the gingival cells. A reduced expression of markers related to stemness, extracellular matrix, and adhesion namely OCT4, NANOG, KRT5, POSTN, COL3A1, CDH1, and CDH3 were seen. The macrophages profoundly affected the production of mineralized nodules by osteoblasts and significantly reduced the expression of COL1A1, OSX, and OCN genes. Conclusion: In periodontitis patients, blood-derived monocytes transform into macrophages of a destructive phenotype due to the characteristic cytokine environment of their GCF. Further, the macrophages affect the genotype and phenotype of the resident cells of the periodontium, aggravate periodontal destruction, as well as jeopardize periodontal healing and resolution of inflammation.
... It can also induce wide neural apoptosis and BBB destruction. Eventually, this damage leads to the inflammatory cascade and aggravates neurovascular dysfunctions [41,164]. Moreover, the massive secretion of inflammatory factors can stimulate the production of the Aβ amyloid protein, while the damaged CECs prevent Aβ-amyloid from being normally cleared, leading to deposition and triggering cerebral amyloid angiopathy [165]. ...
Article
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Vascular dementia (VaD) is the second most common form of dementia worldwide. It is caused by cerebrovascular disease, and patients often show severe impairments of advanced cognitive abilities. Nitric oxide synthase (NOS) and nitric oxide (NO) play vital roles in the pathogenesis of VaD. The functions of NO are determined by its concentration and bioavailability, which are regulated by NOS activity. The activities of different NOS subtypes in the brain are partitioned. Pathologically, endothelial NOS is inactivated, which causes insufficient NO production and aggravates oxidative stress before inducing cerebrovascular endothelial dysfunction, while neuronal NOS is overactive and can produce excessive NO to cause neurotoxicity. Meanwhile, inflammation stimulates the massive expression of inducible NOS, which also produces excessive NO and then induces neuroinflammation. The vicious circle of these kinds of damage having impacts on each other finally leads to VaD. This review summarizes the roles of the NOS/NO pathway in the pathology of VaD and also proposes some potential therapeutic methods that target this pathway in the hope of inspiring novel ideas for VaD therapeutic approaches.
... But what is important about NO features, is its role not only in sepsis development, but also in arterial vasodilatation, bacterial clearance and immune system cells activation (among them e.g. macrophages, neutrophils, T cells) [9]. That affects various organs microperfusion, oxygen delivery and wound healing [10]. ...
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Immunonutrition is one of the most important parts of nutritional treatment in patients with cancer. There are studies which confirm positive effects of using immunonutrition (arginine, glutamine, omega-3 fatty acids, nucleotides, pre- and probiotics) among others on the reduction of the pro-inflammatory cytokines concentrations, shortening of the hospital stay and improvement of the nutritional status. Arginine takes part not only in wound healing process, but also it improves body’s immunity and reduces the incidence of infections. Glutamine reduces the incidence of acute grade 2 and 3 esophagitis and improves quality of life of gastric cancer patients. Omega 3-fatty acids have the ability to inhibit the activity of NF-B. They also reduce the symptoms of graft-versus-host disease in patients undergoing hematopoietic cell transplantation. Nucleotides support the regeneration of intestinal villi. Probiotics play many roles, mainly inhibit the process of carcinogenesis, reduce the incidence of diarrhea and modify intestinal microbiome. However, there are studies indicating the lack of advantages of using immunonutrition compared to standard nutrition. Currently, there is no clear evidence for the use of formulae enriched with immunonutrients versus standard oral nutritional supplements exclusively in the preoperative period. This review summarizes the current knowledge about the role of immunonutrition in supporting treatment of cancer diseases.
... On the basis of the positive results of the three studies focused on NO production in macrophages, radical involved in the modulation of inflammation and immune regulation (40), it might be speculated that edible insect extracts might also have a potential anti-inflammatory activity due to their ability to reduce the release of NO. ...
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Edible insects are proposed as a nutritious and environmentally sustainable alternative source to animal proteins, due to their numerous advantages in terms of reduced ecological impact and high nutritional value. However, the novelty for edible insects relies on the content of bioactive ingredients potentially able to induce a functional effect in the body. The present review summarizes the main findings on the antioxidant properties of edible insects available in the literature. A total of 30 studies involving animals, cell cultures, or in vitro experimental studies evaluating the antioxidant effect of edible insects are presented in this work. When the antioxidant activity was investigated, using a wide variety of in vitro tests and in cellular models, positive results were shown. Dietary supplementation with edible insects was also able to counteract dietary oxidative stress in animal models, restoring the balance of antioxidant enzymes and reducing the formation of oxidation damage markers. On the basis of the reviewed evidences, edible insects might represent a source of novel redox ingredients at low ecological impact able to modulate oxidative stress. However, due to the fact that majority of these evidences have been obtained in vitro and in cellular and animal models, dietary intervention trials are needed to assess the efficacy of edible insect consumption to modulate redox status in humans.
... Nitric oxide (NO) is an agent that functions in various physiological processes, such as immune responses, inflammation, cell deaths, and vascular tone regulation [95]. NO is produced by the enzyme nitric oxide synthetase (NOS). ...
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... In the immune system, NO is usually produced by inducible nitric oxide synthase (iNOS), and its production is transcriptionally induced by upregulation of iNOS in response to cytokines and microbial products (Bogdan, 2015). During microbial pathogenic infection, high output of NO produced by iNOS can dramatically alter microbial metabolism and physiology (MacMicking et al., 1997;Fang, 2004;Lee et al., 2017). ...
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... 2,7,[16][17][18][19][20][21] NO is endogenously generated from the terminal guanidine moiety of L-arginine catalyzed by three different forms of nitric oxide synthases (NOSs), namely neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). 16,22 The constitutive isozymes nNOS and eNOS are expressed in vascular endothelial cells and neurons respectively. 23,24 The third isozyme, iNOS, generated only in response to acute inflammatory stimuli. ...
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Chapter
In the last decades, gasotransmitters have gained attention for their crucial role in pathophysiological and cellular functions. Gasotransmitters are gaseous mediators of the cellular signaling and biological responses from one end to the other. The isoform of these gaseous signaling molecules includes NO (nitric oxide), CO (carbon monoxide), and H2S (hydrogen sulphide), collectively called gasotransmitters. The diverse role of these gasotransmitters in cell and molecular biology as well as biochemical processing has been well validated through several scientific and clinical studies. The biosynthesis, interaction, and movement of gasotransmitters inside the cellular systems are critical especially in terms of their pharmacological response. These gaseous molecules are very toxic and hazardous to human health at higher concentrations but at lower levels they may be considered as therapeutic agents. They can easily diffuse through all the cell membrane and act on their targets for generating pharmacological responses. Due to its gaseous nature, the cellular interactions at the target sites are complex and make it a critical task for researchers to understand. The mode of action and molecular pathways are still under the exploratory phase. Apart from their toxic nature, there are several pharmacological activities such as cardioprotective, antihypertensive, smooth muscle relaxant, vasodilator, antithrombotic, antitumor, etc. that have been reported for these gaseous molecules. This gaseous family has become a promising area for multidisciplinary research in order to establish their importance in relation to disease and health. The perspective of these gasotransmitters is required to validate the clinical translational and future investigation following clear cut understanding of their pharmacological properties. This book chapter deals with the biosynthesis, pharmacological application and clinical utility of gaseous molecules mainly NO and CO.
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We have only recently started to appreciate the extent to which immune cell activation involves significant changes in cellular metabolism. We are now beginning to understand how commitment to specific metabolic pathways influences aspects of cellular biology that are the more usual focus of immunological studies, such as activation‐induced changes in gene transcription, post‐transcriptional regulation of transcription, post‐translational modifications of proteins, cytokine secretion, etc. Here, we focus on metabolic reprogramming in mononuclear phagocytes downstream of stimulation with inflammatory signals (such as LPS and IFNγ) vs alternative activation signals (IL‐4), with an emphasis on work on dendritic cells and macrophages from our laboratory, and related studies from others. We cover aspects of glycolysis and its branching pathways (glycogen synthesis, pentose phosphate, serine synthesis, hexose synthesis, and glycerol 3 phosphate shuttle), the tricarboxylic acid pathway, fatty acid synthesis and oxidation, and mitochondrial biology. Although our understanding of the metabolism of mononuclear phagocytes has progressed significantly over the last 10 years, major challenges remain, including understanding the effects of tissue residence on metabolic programming related to cellular activation, and the translatability of findings from mouse to human biology.
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Introduction: Nitric oxide (NO) is known as the key factor involved in initiating and maintaining an erection. Therefore, NO supplementation may be a target for erectile dysfunction. However, the use of NO donors carries the risk of systemic side effects. Recently, novel NO donors, such as a light-controllable NO donor or NO donor in nanoparticles, have been developed. In this review, we introduce such novel compounds and methods. Aim: To review light-controllable and nanotechnological NO donors for the treatment of erectile dysfunction. Methods: We conducted a review of relevant articles via PubMed in December 2018. Main outcome measures: In this study, we reviewed novel NO donors, such as light-controllable NO donors and nanotechnological NO donors. Results: Some light-controllable NO donors have been already reported. A light-controllable NO donor without metal has also been recently developed. Light-controllable NO donors and light irradiation can control the release of NO spatiotemporally. In an isometric tension study, a relaxing response of the aortic tissue and penile corpus cavernosum was observed under light irradiation with a light-controllable NO donor. In addition, the effects of nanoparticles and nanoemulsions containing sodium nitrate on erectile function have been reported. The nanoformulation containing an NO donor can likely be absorbed percutaneously and, thus, enhance erectile function. Conclusions: A light-controllable NO donor might be useful for treating erectile dysfunction because light irradiation is a convenient method to be applied for patients. However, light permeability might be an issue that needs to be solved. Nanoformulation is also likely to be a useful, non-invasive approach. The application of these procedures and compounds may help in the development of future treatments for erectile dysfunction. Hotta Y, Kataoka T, Taiki Mori T, et al. Review of a Potential Novel Approach for Erectile Dysfunction: Light-Controllable Nitric Oxide Donors and Nanoformulations. Sex Med Rev 2020;8:297-302.
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Nitrous oxide is a common gas used as an anesthetic agent and analgesic medication in operating rooms. The gas inhibits vitamin B12 dependent-methionine synthase, which converts L-homocysteine and 5-methyltetrahydrofolate to L-methionine and tetrahydrofolate, respectively, via a methylation process. The immune system has been frequently reported to be suppressed in anesthetized subjects during the postoperative period. Although previous reviews have focused on the pathophysiologic role of nitrous oxide, none of them has considered the harmful effects of nitrous oxide on the Defense system of the host. In this article, the authors review current studies on the effects of nitrous oxide on the immune system of both patients undergoing surgery and occupational exposure, as well as preclinical studies. Moreover, this paper opens a new horizon for future studies in the context of underlying mechanisms of nitrous oxide actions on the immune system.
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Exhaled Nitric Oxide fraction measurement is a new method for the evaluation of respiratory diseases. It has good correlation with airway inflammation and decreases with the administration of corticosteroids. It is useful as a complement for the diagnosis of asthma, Chronic Obstructive Pulmonary Disease, Cystic Fibrosis and Primary Ciliary Dyskinesia among other respiratory diseases that generate inflammation in the airway. Its assessment is easy, non-invasive, and safe, and the result is obtained immediately. It can be used routinely to evaluate the response and adherence to treatments. This article reviews the biology of Nitric Oxide, and the measurement, interpretation, and main clinical uses of Exhaled Nitric Oxide Fraction.
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Nitric oxide (NO) is a critical transcellular messenger involved in numerous cellular physiology and pathology. Although many electrochemical sensing platforms have been developed for the real-time detection of cellular NO, the dynamic recording of NO release from mechanical deformed cells remains a great challenge. In this communication, we developed a stretchable and transparent NO sensor by spin-coating of silver nanowires (AgNWs) and carbon nanotubes (CNTs) on flexible polydimethylsiloxane (PDMS) substrate. The formed sandwich structure endows the film with robust mechanical flexibility and desirable sensing performance. The developed CNTs/AgNWs/PDMS film exhibited satisfied detection ability toward NO with a wide linear range from 10 nM to 60.94 μM and the detection limit was 2.5 nM. The excellent biocompatibility and mechanical stability of the film provide a suitable environment for cell proliferation and adhesion, enabling the sensing interface to electrochemically detect trace amount of NO released from cells under both static and tensile states in real-time. This research opens a new way for the electrochemical sensing of chemical-signal molecules during mechanotransduction.
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Objectives The study aimed to investigate whether G2/M arrest caused by O2-(2,4-dinitrophenyl) diazeniumdiolate derivative (JS-K) was related to PTEN-mediated inhibition of PI3K/Akt pathway in hepatocellular carcinoma cells. Methods The cell apoptosis was detected by DAPI staining and Annexin V-FITC/PI dual staining. The cell cycle was analysed by PI staining. The expressions of cell cycle-related proteins, PTEN and PI3K/AKT pathway were measured by Western blot. The rat model of primary hepatic carcinoma was established with diethylnitrosamine to verify the antitumour effects of JS-K. Key findings The morphological features of apoptosis were obviously reversed when the cells were pre-treated with bpv(pic), followed by treatment with JS-K. JS-K mediated G2/M arrest and down-regulated expressions of cyclin B1. Meanwhile, it up-regulated the expression of p-Cdk1, p-Chk2 and p-CDC25C while down-regulated that of Cdk1 and CDC25C. Furthermore, JS-K also enhanced the expressions of p21 and p27, PTEN and p53 while decreased the expressions of p-PTEN, PI3K and p-AKT. However, bpv(pic) and Carboxy-PTIO could reverse JS-K-induced G2/M cell arrest and PTEN-mediated inhibition of the PI3K/AKT pathway. The same results were also testified in the rat model of primary hepatic carcinoma. Conclusions JS-K caused G2/M arrest through PTEN-mediated inhibition of the PI3K/AKT pathway involving Chk2/CDC25C/Cdk1 checkpoint.
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The inflammatory process is a mammalian physiological reaction against infectious agents or injuries. Among the cells involved, the macrophages have a highlighted role during this process. Depending on the inflammatory context, they can polarize into pro‐ or anti‐inflammatory profiles (M1 and M2). In this context, compounds derived from cinnamic acid have demonstrated strong evidence of anti‐inflammatory activity; however, the mechanism responsible for this effect remains unclear. In this study, we investigated the anti‐inflammatory activity of five cinnamate‐derived dienes of synthetic origin. The compounds that did not demonstrate significant cytotoxicity were tested to assess anti‐inflammatory activity (NOx) in RAW 264.7 cells stimulated with LPS. Then, the selected compound (diene 1) was evaluated as to its ability to inhibit the secretion of pro‐inflammatory cytokines (IL‐1β, TNF‐α, INF‐γ, MCP‐1, and IL‐6) and increase the production of anti‐inflammatory cytokines (IL‐13, IL‐4, and IL‐10). Finally, diene 1 was able to reduce the expression of TLR4 and increase the phagocytic activity of the macrophages. Gathering these results together, we conclude that diene 1 showed an important anti‐inflammatory effect, and this effect is linked to its immunomodulatory characteristic. Since the M1 markers were reduced at the same time, M2 markers were increased by the treatment of the macrophages with diene 1.
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Objective This study aimed to determine the effects of sacral nerve electrical stimulation (SNS) on neuronal nitric oxide synthase (nNOS) in the colon and sacral cord of rats with defecation disorder after spinal cord injury (SCI). Methods Rats with severe SCI (T10) were used as models and randomly divided into SCI group and SNS group. After 14 days of treatment, the intestinal transport function. Finally, the differences in nNOS immunoreactive cells, protein levels, nNOS mRNA and NO content in the colon and sacral cord tissues were estimated using immunohistochemistry, Western blot, real-time PCR, and nitrate reductase method. Results The intestinal transport function of the SNS group was superior to that of the SCI group (p < 0.05). The AOD of nNOS immunoreactive positive cells in the SCI group were significantly increased compared with those in the sham group. The content of NO of the SCI group significant increased compared with those of the sham group and the SNS group (both p < 0.01). The nNOS mRNA and protein expression was higher in the SCI group than in the sham group (p < 0.05), while that in the SNS group was significantly lower than that in the SCI group. Conclusion SNS could reduce nNOS expression in the colon and sacral cord of SCI rats. This reduction may be an important neuromodulation mechanism for SNS to improve defecation reflex and promote the recovery of intestinal transit function.
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Aim: The aim of this study was to investigate the effect of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced macrophages on head and neck squamous cell carcinoma (HNSCC) cell line proliferation and invasion. Main methods: THP-1 monocytes were differentiated toward macrophages using 12.5 ng/ml phorbol 12-myristate 13-acetate treatment for 48 h. The expression of interleukin-6 (IL-6) mRNA and cytokine by monocytes and macrophages was determined using real time PCR and ELISA, respectively. The cells were analyzed for CD14 expression using immunofluorescent labeling. The macrophages were induced using 1 μg/ml P. gingivalis LPS for 24 h, and the conditioned medium (CM) was collected. The monocyte, macrophage, and LPS-induced macrophage CM were evaluated for IL-6 and tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO) content using ELISA and the Griess Reagent System, respectively. Human primary (HN18, HN30, and HN4) and metastatic (HN17, HN31, and HN12) HNSCC cell lines were treated with the monocyte, macrophage, and LPS-induced macrophage CM. The proliferation and invasion of the HNSCC cell lines were evaluated using MTT and modified Boyden chamber assays, respectively. Key findings: Macrophages demonstrated increased IL-6 and CD14 expression. The P. gingivalis LPS significantly induced macrophage NO secretion, however, that of TNF-α decreased. The LPS-induced macrophages CM inhibited HN4 proliferation. Interestingly, the LPS-induced macrophage CM promoted invasion of all HNSCC cell lines. Significance: Our data demonstrate that P. gingivalis LPS-induced macrophages increased NO secretion. The activated macrophage CM inhibited HN4 cell proliferation and promoted invasion of all HNSCC cell lines.
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4-(Nitrophenyl)hydrazone derivatives of N-acylhydrazone were synthesized and screened for suppress lymphocyte proliferation and nitrite inhibition in macrophages. Compared to an unsubstituted N-acylhydrazone, active compounds were identified within initial series when hydroxyl, chloride and nitro substituents were employed. Structure-activity relationship was further developed by varying the position of these substituents as well as attaching structurally-related substituents. Changing substituent position revealed a more promising compound series of anti-inflammatory agents. In contrast, an N-methyl group appended to the 4-(nitrophenyl)hydrazone moiety reduced activity. Anti-inflammatory activity of compounds is achieved by modulating IL-1β secretion and prostaglandin E2 synthesis in macrophages and by inhibiting calcineurin phosphatase activity in lymphocytes. Compound SintMed65 was advanced into an acute model of peritonitis in mice, where it inhibited the neutrophil infiltration after being orally administered. In summary, we demonstrated in great details the structural requirements and the underlying mechanism for anti-inflammatory activity of a new family of hydrazone-N-acylhydrazone, which may represent a valuable medicinal chemistry direction for the anti-inflammatory drug development in general.
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Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage- mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase- polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell- permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.
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In rheumatoid arthritis (RA), nitric oxide (NO) is implicated in inflammation, angiogenesis and tissue destruction. The enzyme inducible nitric oxide synthase (iNOS) is responsible for the localised overproduction of NO in the synovial joints affected by RA. The pro-and anti-inflammatory cytokines stimulate the synovial macrophages and the fibroblast-like synovio-cytes to express iNOS. Therefore, the cytokine signalling network underlying the regulation of iNOS is essential to understand the pathophysiology of the disease. By using information from the literature, we have constructed, for the first time, the cytokine signalling network involved in the regulation of iNOS expression. Using the differential expression patterns obtained by re-analysing the microarray data on the RA synovium and the synovial macro-phages available in the Gene Expression Omnibus (GEO) database, we aimed to establish the role played by the network genes towards iNOS regulation in the RA synovium. Our analysis reveals that the network genes belonging to interferon (IFN) and interleukin-10 (IL-10) pathways are always up-regulated in the RA synovium whereas the genes which are part of the anti-inflammatory transforming growth factor-beta (TGF-β) signalling pathway are mostly down-regulated. We observed a consistent up-regulation of the transcription factor signal transducers and activators of transcription 1 (STAT1) in the RA synovium and the macrophages. Interestingly, we found a consistent up-regulation of the iNOS interacting protein ras-related C3 botulinum toxin substrate 2 (RAC2) in the RA synovium as well as the macrophages. Importantly, we have constructed a model to explain the impact of IFN and IL-10 pathways on Rac2-iNOS interaction leading to overproduction of NO and thereby causing chronic inflammation in the RA synovium. The interplay between STAT1 and RAC2 in the regulation of NO could have implications for the identification of therapeutic targets for RA.
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The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa Bd/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS treated mouse macrophages.
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p300, a ubiquitous transcription coactivator, plays an important role in gene activation. Our previous work demonstrated that human inducible nitric oxide synthase (hiNOS) expression can be highly induced with the cytokine mixture (CM) of TNF-α + IL-1β + IFN-γ. In this study, we investigated the functional role of p300 in the regulation of hiNOS gene expression. Our initial data showed that overexpression of p300 significantly increased the basal and cytokine-induced hiNOS promoter activities in A549 cells. Interestingly, p300 activated cytokine-induced hiNOS transcriptional activity was completely abrogated by deleting the upstream hiNOS enhancer at -5 kb to -6 kb in the promoter. Furthermore, p300 over-expression increased cytokine-induced transcriptional activity on a heterologous minimal TK promoter with the same hiNOS enhancer. Site-directed mutagenesis of the hiNOS AP-1 motifs revealed that an intact upstream (-5.3kb) AP-1 binding site was critical for p300 mediated cytokine-induced hiNOS transcription. Furthermore, our ChIP analysis demonstrated that p300 was binding to Jun D and Fra-2 proteins at -5.3 kb AP-1 binding site in vivo. Lastly, our 3C assay was able to detect a long DNA loop between the hiNOS enhancer and core promoter site, and ChIP loop assay confirmed that p300 binds to AP-1 and RNA pol II proteins. Overall, our results suggest that coactivator p300 mediates cytokine-induced hiNOS transactivation by forming a distant DNA loop between its enhancer and core promoter region.
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Nitric oxide, a multifunctional effector molecule syn- thesized by nitric oxide synthase (NOS) from L-arginine, conveys signals for vasorelaxation, neurotransmission, and cytotoxicity. Three different NOS isoforms have been identified which fall into two distinct types, con- stitutive and inducible. The inducible NOS (iNOS) iso- form is expressed in a variety of cell types and tissues in response to inflammatory agents and cytokines. The hu- man MOS (NOS2) gene was isolated on overlapping cos- mid clones &om a human genomic library using both the murine macrophage and the human hepatocyte iNOS cDNAs as probes. All isolated cosmids were part of a single genomic locus and no other genomic loci were identified or isolated. Analysis of this locus indicated that the human iNOS gene is -37 kilobases in length and consists of 26 exons and 25 introns. Primer extension analysis of lipopolysaccharide and cytokine-stimulated human hepatocyte RNA mapped the transcriptional ini- tiation site 30 base pairs downstream of a TATA se- quence, and a 400-base pair S’-flanking region was found to be structurally similar to the recently described mu- rine iNOS promoter. Polymerase chain reaction analysis of a humadrodent genomic DNA somatic cell hybrid panel and fluorescent in situ hybridization indicated that the human iNOS gene is located on chromosome 17 at position 17cen-q11.2
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Oxidized low-density lipoprotein (ox-LDL) is assumed to be a major causal agent in hypercholesteraemia-induced atherosclerosis. Because the proliferation of lipid-loaden macrophages within atherosclerotic lesions has been described, we investigated the dependence of macrophage proliferation on the inhibition of inducible nitric oxide synthase (iNOS) by hypochlorite oxidized LDL. Ox-LDL induces a dose dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-interferon stimulated mouse macrophages (J774.A1) with concomitant macrophage proliferation as assayed by cell counting, tritiated-thymidine incorporation and measurement of cell protein. Native LDL did not influence macrophage proliferation and inducible nitric oxide synthesis. iNOS protein and mRNA was reduced by HOCl-oxidized LDL (0-40 ng/ml) as revealed by immunoblotting and competitive semiquantitative PCR. Macrophage proliferation was increased by the addition of the iNOS inhibitor L-NAME. The addition of ox-LDL to L-NAME containing incubations induced no further statistically significant increase in cell number. Nitric oxide donors decreased ox-LDL induced macrophage proliferation and nitric oxide scavengers restored macrophage proliferation to the initial values achieved by ox-LDL. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with ox-LDL demonstrates that the proliferative actions of ox-LDL are associated with a decrease of NO-induced apoptosis. Our data show that inhibition of iN-OS dependent nitric oxide production caused by hypochlorite oxidized LDL enhances macrophage proliferation. This might be a key event in the pathogenesis of atherosclerotic lesions.
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The ability of certain species of Chlamydia to inhibit the biogenesis of phagolysosomes permits their survival and replication within macrophages. Survival of macrophage-adapted chlamydiae correlates with the multiplicity of infection (MOI), and optimal chlamydial growth occurs in macrophages infected at MOI ≤ 1. In this study, we examined the replicative capacity of Chlamydia muridarum in the RAW 264.7 murine macrophage cell line at different MOI. C. muridarum productively infected these macrophages at low MOI but yielded few viable EB when macrophages were infected at moderate (10) or high (100) MOI. While high MOI caused cytotoxicity and irreversible host cell death, macrophages infected at a moderate MOI did not show signs of cytotoxicity until late in the infectious cycle. Inhibition of host protein synthesis rescued C. muridarum in macrophages infected at a moderate MOI, implying that chlamydial growth was blocked by activated defense mechanisms. Conditioned medium from these macrophages was anti-chlamydial and contained elevated levels of IL-1β, IL-6, IL-10 and IFN-β. Macrophage activation depended on TLR2 signaling and cytokine production required live, transcriptionally active chlamydiae. A hydroxyl radical scavenger and inhibitors of iNOS and cathepsin B also reversed chlamydial killing. High levels of reactive oxygen species (ROS) led to an increase in cathepsin B activity, and pharmacological inhibition of ROS and cathepsin B reduced iNOS expression. Our data demonstrate that MOI dependent TLR2 activation of macrophages results in iNOS induction via a novel ROS and cathepsin-dependent mechanism to facilitate C. muridarum clearance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Upon their activation, CD4 T cells can differentiate into distinct T helper cell subsets with specialised functions. Different T helper cell subsets produce specific cytokines that mediate beneficial and sometimes detrimental effects, depending on the infection or disease setting. CD4 T cell priming relies on signals delivered by the T cell antigen receptor, co-stimulatory receptors and cytokine receptors on the CD4 T cell surface. Cytokine receptors are well known to deliver instructive signals that direct T helper cell differentiation. However, it is less appreciated that co-stimulatory receptors also exert potent modulatory effects on this process. In this review, we outline the contribution of co-stimulatory and co-inhibitory receptors to the process of T helper cell differentiation, focusing on those pathways for which the underlying mechanisms are best known. Herein, we depict the physiological context of T cell priming and emphasise the impact of cell-cell communication on directing T helper cell differentiation.Immunology and Cell Biology accepted article preview online, 24 March 2015. doi:10.1038/icb.2015.45.
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Background Accumulation of nitrated protein is seen in peripheral lung and cells from patients with chronic obstructive pulmonary disease (COPD). Nitrated protein causes abnormal protein function, but the nitration was believed to be an irreversible process. However, there are accumulating evidences that this process is reversible by an active denitration pathway. The aim of this study is to detect denitration activity in protein extracts from peripheral lung tissue of COPD and to compare with those in healthy subjects. Materials and Methods Peripheral lung tissue from 4 healthy, 4 smokers without COPD, 4 GOLD stage 1 and 4 GOLD stage 2 were used for denitration assay. Denitration activity was determined as reduction of nitro-tyrosine level of nitrated histone protein after incubation with protein extracts from peripheral lung, which was determined by western blotting. In addition, RNA is extracted from peripheral lung of 8 healthy, 7 smoking control, 8 stage 1 and 2 COPD and 10 stage 3 and 4 COPD and nitrate reductase mRNA expression was determined by real time RT-PCR. Results Peripheral lung protein extracts from healthy subjects reduced nitro-tyrosine level of nitrated histone. Thus, we were able to show denitration activity in peripheral lungs. The denitration activity was slightly reduced in smoking controls, and significantly reduced in COPD patients. We also showed that the expression of the human homologue of nitrate reductase (chytochrome β2 reductase), a potential candidate of denitrase, was significanty reduced in COPD lung. Conclusion This study suggests that accumulation of nitrated protein in lung tissue of COPD may, at least in part, be induced by a reduction in denitration activity or nitrate reductase.
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The inducible form of nitric oxide synthase (iNOS) is expressed during inflammation of the intestine and may contribute to tissue injury. We have examined iNOS transcriptional regulation in DLD-1 cells, a human intestinal epithelial line that produces large amounts of nitric oxide and iNOS mRNA in response to a combination of the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Levels of iNOS mRNA are extremely low in unstimulated DLD-1 cells but increase dramatically after cytokine treatment. Nuclear run-on analyses demonstrated that transcriptional activation, which accounts for a portion of this increase, is dependent on both IL-1 beta and IFN-gamma and requires de novo protein synthesis. Transfection of DLD-1 cells with reporter constructs containing deletions of the iNOS promoter showed that sequences located between 8.7 and 10.7 kb upstream of the transcription initiation site are necessary for cytokine responsiveness. This region contains potential binding sites for several cytokine-induced transcription factors and was shown to function in either orientation when placed upstream of a basal iNOS promoter segment terminating at-1.1 kb. The extremely distal location of the cytokine-responsive region contrasts with the reported positions of elements involved in the regulation of iNOS transcription in other cell types. Our data also suggest that posttranscriptional events could play a significant role in regulating iNOS gene expression in human intestinal epithelia.
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Atherosclerosis is a leading cause of morbidity and mortality in Western society. Despite improved insight into disease pathogenesis and therapeutic options, additional treatment strategies are required. Emerging evidence highlights the relevance of endothelial cell (EC) metabolism for angiogenesis, and indicates that EC metabolism is perturbed when ECs become dysfunctional to promote atherogenesis. In this review, we overview the latest insights on EC metabolism and discuss current knowledge on how atherosclerosis deregulates EC metabolism, and how maladaptation of deregulated EC metabolism can contribute to atherosclerosis progression. We will also highlight possible therapeutic avenues, based on targeting EC metabolism.
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