Overexpression Of Nitric Oxide Synthase Research Articles

Analgesic Effects of Two Types of Spinal Manipulation in Acute Lumbar Radiculopathy Model Rats.

To compare the analgesic effects of two types of spinal manipulation (SM) in acute lumbar radiculopathy (ALR) model rats induced by self-transplantation of autologous nucleus pulposus (ANP), and clarify the therapeutic mechanism. Totally 108 male Sprague-Dawley rats were randomly divided into 6 groups by a random number table (18 rats in each group), including a blank group with no interference, a sham operation group with a surgery by making a local soft tissue incision on the left side of L5-6 vertebral segment, a model group with ALR of L5 extraforaminal nerve by ANP self-transplantation without other interference, a sham manipulation (SMA) group with simulating physical rotation, as well as a mobilization (MOB) group with simulating low-velocity and variable-amplitude rotation and a manipulation (MAN) group with simulating high-velocity and low-amplitude rotation. The interventions in SMA, MOB, and MAN groups started 1 day after modeling followed by another 5 treatments at days 3, 5, 8, 10 and 12. Rats in the other 3 groups did not receive any special intervention. Behavioral pain tests of 50% mechanical pain withdrawal threshold (50% PWT) and paw withdrawal latency (PWL) were conducted 1 day before operation followed by another 10 tests on days 1-7, 10, 12 and 14. Immunohistochemical expression of nitric oxide synthase (NOS) was investigated on days 5 and 12 after operation. After 3 experimental SM interventions, 50% PWT and PWL were higher in the MAN group than the SMA group on days 6 and 7, and higher on days 10, 12 and 14 postoperatively (P<0.05 or P<0.01), while the same indices were significantly higher in the MOB group than MAN group on days 1-4 (P<0.05 or P<0.01). The expression of NOS was lower in the MAN and MOB groups than SMA group on day 12 postoperatively (P<0.01). Both manipulation and mobilization produced better results than sham interference in relieving pain by reducing neuroinflammation possibly. At the early period, compared with manipulation, mobilization presented less sensitive response to pain until later visit. SM may inhibit the overexpression of NOS, thereby alleviating severe radiculopathy.

Sex-Specific Regulation of Interferon-γ Cytotoxicity in Mouse Liver by Autophagy.

Interferon-γ (IFNγ) is a central activator of immune responses in the liver and other organs. IFNγ triggers tissue injury and inflammation in immune diseases, which occur predominantly in females for unknown reasons. Recent findings that autophagy regulates hepatotoxicity from proinflammatory cytokines led to an examination of whether defective hepatocyte autophagy underlies sex-specific liver injury and inflammation induced by IFNγ. A lentiviral autophagy-related 5 (Atg5) knockdown was performed to decrease autophagy-sensitized alpha mouse liver (AML 12) hepatocytes to death from IFNγ in combination with IL-1β or TNF. Death was necrosis attributable to impaired energy homeostasis and adenosine triphosphate depletion. Male mice with decreased autophagy from a tamoxifen-inducible, hepatocyte-specific Atg5 knockout were resistant to IFNγ hepatotoxicity whereas female knockout mice developed liver injury and inflammation. Female mice had increased IFNγ-induced signal transducer and activator of transcription 1 (STAT1) levels compared to males. Blocking STAT1, but not interferon regulatory factor 1, signaling prevented IFNγ-induced hepatocyte death in autophagy-deficient AML12 cells and female mice. The mechanism of death is STAT1-induced overexpression of nitric oxide synthase 2 (NOS2) as in vitro hepatocyte death and in vivo liver injury were blocked by NOS2 inhibition. Decreased hepatocyte autophagy sensitizes mice to IFNγ-induced liver injury and inflammation through overactivation of STAT1 signaling that causes NOS2 overexpression. Hepatotoxicity is restricted to female mice, suggesting that sex-specific effects of defective autophagy may underlie the increased susceptibility of females to IFNγ-mediated immune diseases.

Effects of endogenous H2S production inhibition on the homeostatic responses induced by acute high-salt diet consumption.

The gaseous modulator hydrogen sulfide (H2S) is synthesized, among other routes, by the action of cystathionine-γ-lyase (CSE) and importantly participates in body fluid homeostasis. Therefore, the present study aimed to evaluate the participation of H2S in behavioral, renal and neuroendocrine homeostatic responses triggered by the acute consumption of a high Na+ diet. After habituation, adult male Wistar rats were randomly distributed and maintained for seven days on a control [CD (0.27% of Na+)] or hypersodic diet [HD (0.81% of Na+)]. CD and HD-fed animals were treated with DL-Propargylglycine (PAG, 25mg/kg/day, ip) or vehicle (0.9% NaCl in equivalent volume) for the same period. At the end of the experiment, animals were euthanized for blood and tissue collection. We demonstrated that a short-term increase in dietary Na+ intake, in values that mimic the variations in human consumption (two times the recommended) significantly modified hydroelectrolytic homeostasis, with repercussions in the hypothalamic-neurohypophysial system and hypothalamic-pituitary-adrenal axis function. These findings were accompanied by the development of a clear inflammatory response in renal tubular cells and microvascular components. On the other hand, the inhibition of the endogenous production of H2S by CSE provided by PAG treatment prevented the inflammation induced by HD. In the kidney, PAG treatment induced the overexpression of inducible nitric oxide synthase in animals fed with HD. Taken together, these data suggest, therefore, that HD-inducedH2S productionplays an important proinflammatory role in the kidney, apparently counter regulating nitric oxide actions in renal tissue.

Anti-Inflammatory Effects and Their Correlation with Microbial Community of Shindari, a Traditional Jeju Beverage

In this study, we investigated the microbial community and its correlation with anti-inflammatory effects during the fermentation procedure of shindari. Since shindari is traditionally manufactured through a fermentation process of 3–4 days in summer or 5–6 days in winter, the shindari materials applied in this study were fermented for 1, 3, and 5 days, respectively. Microbial community structure in the shindari manufacturing process was analyzed by Illumina MiSeq sequencing. Results revealed different fermentation time had different influences on the community structure and microbial diversity in the shindari process. Ultimately, we found that pathogenic bacteria such as Enterococcus durans (9.3%) and Enterobacter asburiae (4.5%) dominate the shindari fermented for 1 day, while samples with a fermentation period of more than 3 days occupied lactic acid bacteria such as Pediococcus sp. (97.1%). In addition, to study the relationship between fermentation period and anti-inflammatory effect in the shindari manufacturing process, we applied RAW264.7 cells, a classic cell model for inflammation studies. First, we prepared an ethyl acetate extract of shindari fermented for 1 (S1), 3 (S3), or 5 days (S5), and then it was confirmed that all of these extracts inhibited the nitric oxide (NO) production in a concentration-dependent manner. In addition, these inhibitory effects were correlated with the suppressive effect of shindari extracts against overexpression of inducible nitric oxide synthase (iNOS). Furthermore, S3 and S5 also inhibited the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. Overall, the anti-inflammatory effect of S3 was suggested to be mediated through the negative regulation of mitogen-activated protein kinase signaling (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways.

Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

Nitric Oxide Participates in the Brain Ischemic Tolerance Induced by Intermittent Hypobaric Hypoxia in the Hippocampal CA1 Subfield in Rats.

Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH + sham group, ischemia group and IH + ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000m high-altitude (PB = 404mmHg, PO2 = 84mmHg) 6h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, L-NAME + ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of L-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.

Optimizing endothelial cell functionalization for cell therapy of vascular proliferative disease using a direct contact co-culture system.

Increased susceptibility to thrombosis, neoatherosclerosis, and restenosis due to incomplete regrowth of the protective endothelial layer remains a critical limitation of the interventional strategies currently used clinically to relieve atherosclerotic obstruction. Rapid recovery of endothelium holds promise for both preventing the thrombotic events and reducing post-angioplasty restenosis, providing the rationale for developing cell delivery strategies for accelerating arterial reendothelialization. The successful translation of experimental cell therapies into clinically viable treatment modalities for restoring vascular endothelium critically depends on identifying strategies for enhancing the functionality of endothelial cells (EC) derived from high cardiovascular risk patients, the target group for the majority of angioplasty procedures. Enhancing EC-associated nitric oxide (NO) synthesis by inducing overexpression of NO synthase (NOS) has shown promise as a way of increasing paracrine activity and restoring function of EC. In the present study, we developed a direct contact co-culture approach compatible with highly labile effectors, such as NO, and applied it for determining the effect of EC functionalization via NOS gene transfer on the growth of co-cultured arterial smooth muscle cells (A10 cell line) exhibiting the defining characteristics of neointimal cells. Bovine aortic endothelial cells magnetically transduced with inducible NOS-encoding adenovirus (Ad) formulated in zinc oleate-based magnetic nanoparticles (MNP[iNOSAd]) strongly suppressed growth of proliferating A10 and attenuated the stimulatory effect of a potent mitogen, platelet-derived growth factor (PDGF-BB), whereas EC functionalization with free iNOSAd or MNP formulated with a different isoform of the enzyme, endothelial NOS, was associated with lower levels of NO synthesis and less pronounced antiproliferative activity toward co-cultured A10 cells. These results show feasibility of applying magnetically facilitated gene transfer to potentiate therapeutically relevant effects of EC for targeted cell therapy of restenosis. The direct contact co-culture methodology provides a sensitive and reliable tool with potential utility for a variety of biomedical applications.

DNA Double-Strand Breaks Caused by Different Microorganisms: A Special Focus on Helicobacter pylori.

The association between inflammation and cancer has long been recognized. Several studies have found that different types of tumors develop at sites of chronic inflammation. It is stated that over 15%-20% of malignancies worldwide can be related to infections caused by viruses, bacteria, and schistosomes. Inflammatory conditions are characterized by overexpression of inducible nitric oxide synthase (iNOS) and overproduction of nitric oxide/reactive nitrogen species (ROSs/RNSs) in epithelial cells. Reactive oxygen species (ROSs) may also lead to cellular alterations and eventually to inflammation. A variety of chronic infectious diseases can generate steady-state levels of ROSs/RNSs within infected cells and possibly lead to different types of DNA lesions. Accumulation of DNA lesions may finally lead to mutations that may activate oncogenes or inactivate tumor suppressor genes. Helicobacter pylori has been shown to generate ROSs/RNSs, induce DNA damage, and lead to chronic inflammation in gastric epithelial cells. A limited number of studies have addressed the effects of Helicobacter pylori on DNA damage, particularly its impact on single-strand and double-strand DNA breaks. This bacterium is classified as a Group I carcinogen by the International Agency for Research on Cancer on the basis of numerous animal and epidemiological studies. Chronic Helicobacter pylori infection can lead to increased risk of gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. This review addresses the DNA-damaging and double-strand break-inducing effects of different microorganisms and their toxins, specifically focusing on Helicobacter pylori.

Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1.

Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington’s disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein in vivo.

Abstract 4317: Black raspberries show potent activity in prevention of experimental squamous cell esophageal cancer compared to a combination of selective COX-2 and iNOS inhibitors

Abstract Esophageal squamous cell carcinoma (SCC) remains a major health threat worldwide and preventive strategies are needed. We previously reported that overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are correlated with nitrosamine-induced squamous carcinogenesis of the esophagus and the selective iNOS and COX-2 inhibitors significantly inhibit incidence and progression. We also demonstrated anti-cancer activity of black raspberries (BRB) in a rodent model of esophageal SCC. The objective of the current study was to compare the impact of BRB versus the combination of celecoxib, a selective COX-2 inhibitor, and S,S’-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), a selective iNOS inhibitor, in inhibition of SCC of the esophagus, and to elucidate molecular mechanism of an effective cancer prevention. We found that BRB is superior to the combination of two drugs in suppression of premalignant tissue growth in the rat esophagi. Moreover, rats fed BRB have lower tumor multiplicity than those fed celcecoxib + PBIT. Our data indicated that BRB also shows a potent inhibitory effect on esophageal iNOS and COX-2, in addition to modulating several associated oncogenic cell signaling pathways. We further conducted parallel mechanistic studies in vitro using BRB anthocyanins and the above two drugs. Our findings demonstrated that dietary BRB is superior to the combination of two chemopreventive pharmaceutical agents in prevention of experimental squamous cell esophageal cancer, suggesting the potential value of additional translational studies in developing food-based products using BRB for the prevention of esophageal SCC carcinogenesis in humans (Supported by NIH/NCI R01 CA131073). Citation Format: Ni Shi, Kenneth M. Riedl, Steven J. Schwartz, Xiaoli Zhang, Steven K. Steven, Tong Chen. Black raspberries show potent activity in prevention of experimental squamous cell esophageal cancer compared to a combination of selective COX-2 and iNOS inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4317.

Production and physiological role of NO in the oral cavity

SummaryNitric oxide (NO) is a free radical which is produced from a wide variety of cells and tissues in the human body. NO is involved in the regulation of many physiological processes, such as vascular relaxation, neurotransmission, immune regulation, and cell death. NO is generated by nitric oxide synthase (NOS), which has three identified isoforms: neuronal type NOS (nNOS), endothelial type NOS (eNOS), and inducible type NOS (iNOS). Different isoforms are expressed depending on the organs, tissues, and cells, and investigation of the types and functions of enzymes expressed in various tissues is underway. The oral cavity is a space in which marked changes have been detected in NO levels, and each tissue is constantly influenced by NO. NO is a component of saliva and is produced by oral bacteria in the oral cavity and released by NOS expressed in oral mucosa. NOS isoforms expressed under normal conditions differ among the oral organs. In addition, the overexpression of NOS was involved in carcinogenesis and tumor growth progression. This review summarized the expression of NOS and functions of NO in oral cavity organs, and their roles in diseases and the influences of treatments.

Unchanged NADPH Oxidase Activity in Nox1-Nox2-Nox4 Triple Knockout Mice: What Do NADPH-Stimulated Chemiluminescence Assays Really Detect?

NADPH oxidases of the Nox family are considered important sources of cellular reactive oxygen species (ROS) production. This conclusion is, in part, based on the ability of NADPH to elicit a chemiluminescence signal in tissue/cell homogenates or membrane preparations in the presence of enhancers such as lucigenin, luminol, or L012. However, the ability of these particular assays to specifically detect Nox activity and Nox-derived ROS has not been proven. In this study, we demonstrate that combined knockout of the three main Nox enzymes of the mouse (Nox1-Nox2-Nox4 triple knockout) had no impact on NADPH-stimulated chemiluminescence signals in the aorta, heart, and kidney homogenates. In the NADPH-stimulated membrane assays, no effect of in vivo angiotensin II pretreatment or deletion of Nox enzymes was observed. In in vitro studies in HEK293 cells, the overexpression of Nox5 or Nox4 markedly increased ROS production in intact cells, whereas overexpression of Nox5 or Nox4 had no influence on the signal in membrane assays. In contrast, overexpression of nitric oxide synthase or cytochrome P450 enzymes resulted in an increased chemiluminescence signal in isolated membranes. On the basis of these observations, we propose the hypothesis that NADPH-stimulated chemiluminescence-based membrane assays, as currently used, do not reflect Nox activity.

M2 Macrophage Polarization Mediates Anti-inflammatory Effects of Endothelial Nitric Oxide Signaling.

Endothelial nitric oxide (NO) signaling plays a physiological role in limiting obesity-associated insulin resistance and inflammation. This study was undertaken to investigate whether this NO effect involves polarization of macrophages toward an anti-inflammatory M2 phenotype. Mice with transgenic endothelial NO synthase overexpression were protected against high-fat diet (HFD)-induced hepatic inflammation and insulin resistance, and this effect was associated with reduced proinflammatory M1 and increased anti-inflammatory M2 activation of Kupffer cells. In cell culture studies, exposure of macrophages to endothelial NO similarly reduced inflammatory (M1) and increased anti-inflammatory (M2) gene expression. Similar effects were induced by macrophage overexpression of vasodilator-stimulated phosphoprotein (VASP), a key downstream mediator of intracellular NO signaling. Conversely, VASP deficiency induced proinflammatory M1 macrophage activation, and the transplantation of bone marrow from VASP-deficient donor mice into normal recipients caused hepatic inflammation and insulin resistance resembling that induced in normal mice by consumption of an HFD. These data suggest that proinflammatory macrophage M1 activation and macrophage-mediated inflammation are tonically inhibited by NO → VASP signal transduction, and that reduced NO → VASP signaling is involved in the effect of HFD feeding to induce M1 activation of Kupffer cells and associated hepatic inflammation. Our data implicate endothelial NO → VASP signaling as a physiological determinant of macrophage polarization and show that signaling via this pathway is required to prevent hepatic inflammation and insulin resistance.

Enhanced expression of hypothalamic nitric oxide synthase in rats developmentally exposed to organophosphates

Nitric oxide synthase (NOS) is highly expressed in the hypothalamus, and nitric oxide (NO) specifically contributes to the regulation of neuronal activity within distinct hypothalamic regions. We studied the long-lasting effects of developmental exposure to low doses of organophosphate chlorpyrifos (CPF) and diazinon (DZN) on the expression of NOS in the hypothalamic subnuclei that subserve neuroendocrine, autonomic and cognitive functions. A daily dose of 1mg/kg of either CPF or DZN was administered to developing rats during gestational days 15–18 or postnatal days (PND) 1–4. Brain sections from PND 60 rats were processed using NADPH-diaphorase (NADPH-d) and neuronal NOS (nNOS) immunohistochemistry. The number of labeled neurons and the optical density (OD) were assessed in the supraoptic (SON), paraventricular (PVN), medial septum, vertical limb, and horizontal limb of the diagonal band. Developmental exposure to organophosphates increased the number of labeled neurons and OD in different subnuclei in the hypothalamus without gender selectivity. The effect on OD was more pronounced and was significant for more cases. Prenatal exposure to CPF and DZN significantly increased the OD in all regions studied with the exception of PVN. Neonatal exposure to DZN also consistently increased OD in all studied subnuclei. For rats that treated with CPF during early postnatal period, this effect was statistically significant only for the SON and PVN. These findings suggest that overexpression of NOS in the hypothalamus may contribute to the mechanisms inducing or compensating for endocrine, autonomic and cognitive abnormalities after developmental exposure to organophosphates.

Exercise training in adverse cardiac remodeling

Cardiac remodeling in response to a myocardial infarction or chronic pressure-overload is an independent risk factor for the development of heart failure. In contrast, cardiac remodeling produced by regular physical exercise is associated with a decreased risk for heart failure. There is evidence that exercise training has a beneficial effect on disease progression and survival in patients with cardiac remodeling and dysfunction, but concern has also been expressed that exercise training may aggravate pathological remodeling and dysfunction. Here we present studies from our laboratory into the effects of exercise training on pathological cardiac remodeling and dysfunction in mice. The results indicate that even in the presence of a large infarct, exercise training exerts beneficial effects on the heart. These effects were mimicked in part by endothelial nitric oxide synthase (eNOS) overexpression and abrogated by eNOS deficiency, demonstrating the importance of nitric oxide signaling in mediating the cardiac effects of exercise. Exercise prior to a myocardial infarction was also cardioprotective. In contrast, exercise tended to aggravate pathological cardiac remodeling and dysfunction in the setting of pressure-overload produced by an aortic stenosis. These observations emphasize the critical importance of the underlying pathological stimulus for cardiac hypertrophy and remodeling, in determining the effects of exercise training. Future studies are needed to define the influence of exercise type, intensity and duration in different models and severities of pathological cardiac remodeling. Together such studies will aid in optimizing the therapy of exercise training in the setting of cardiovascular disease.

Nitric Oxide Synthase-3 Promotes Embryonic Development of Atrioventricular Valves

Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3−/− mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3−/− compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3−/− mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1+ cells in the AV cushion were decreased in NOS3−/− compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ), bone morphogenetic protein (BMP2) and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3−/− compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.

Nitric oxide synthase-3 deficiency results in hypoplastic coronary arteries and postnatal myocardial infarction

Hypoplastic coronary artery disease is a rare congenital abnormality that is associated with sudden cardiac death. However, molecular mechanisms responsible for this disease are not clear. The aim of the present study was to assess the role of nitric oxide synthase-3 (NOS3) in the pathogenesis of hypoplastic coronary arteries. Wild-type (WT), NOS3(-/-), and a novel cardiac-specific NOS3 overexpression mouse model were employed. Deficiency in NOS3 resulted in coronary artery hypoplasia in foetal mice and spontaneous myocardial infarction in postnatal hearts. Coronary artery diameters, vessel density, and volume were significantly decreased in NOS3(-/-) mice at postnatal day 0. In addition, NOS3(-/-) mice showed a significant increase in the ventricular wall thickness, myocardial volume, and cardiomyocyte cell size compared with WT mice. Lack of NOS3 also down-regulated the expression of Gata4, Wilms tumour-1, vascular endothelial growth factor, basic fibroblast growth factor and erythropoietin, and inhibited migration of epicardial cells. These abnormalities and hypoplastic coronary arteries in the NOS3(-/-) mice were completely rescued by the cardiac-specific overexpression of NOS3. Nitric oxide synthase-3 is required for coronary artery development and deficiency in NOS3 leads to hypoplastic coronary arteries.

Targeting Hepatoma Using Nitric Oxide Donor Strategies

The study evaluated the role of increased intracellular nitric oxide (NO) concentration using NO donors or stably NO synthase-3 (NOS-3) overexpression during CD95-dependent cell death in hepatoma cells. The expression of cell death receptors and caspase activation, RhoA kinase activity, NOS-3 expression/activity, oxidative/nitrosative stress, and p53 expression were analyzed. The antitumoral activity of NO was also evaluated in the subcutaneous implantation of NOS-3-overexpressing hepatoma cells, as well NO donor injection into wild-type hepatoma-derived tumors implanted in xenograft mouse models. NO donor increased CD95 expression and activation of caspase-8 and 3 in HepG2, Huh7, and Hep3B cells. NOS-3 overexpression increased oxidative/nitrosative stress, p53 and CD95 expression, cellular Fas-associated death domain (FADD)-like IL-1beta converting enzyme (FLICE) inhibitory protein long (cFLIP(L)) and its short isoform (cFLIP(S)) shift, and cell death in HepG2 (4TO-NOS) cells. The inhibition of RhoA kinase and p53 knockdown using RNA interference reduced cell death in 4TO-NOS cells. The supplementation with hydrogen peroxide (H(2)O(2)) increased NOS-3 activity and cell death in 4TO-NOS cells. NOS-3 overexpression or NO donor injection into hepatoma-derived tumors reduced the size and increased p53 and cell death receptor expression in nude mice. The increase of intracellular NO concentration promoted oxidative and nitrosative stress, Rho kinase activity, p53 and CD95 expression, and cell death in cultured hepatoma cells. NOS-3-overexpressed HepG2 cells or intratumoral NO donor administration reduced tumor cell growth and increased the expression of p53 and cell death receptors in tumors developed in a xenograft mouse model.

Mitochondria Specific Antioxidants and their Derivatives in the Context of the Drug Development for Neurodegeneration and Cancer

Neurodegeneration and cancer are fast becoming the leading causes of age-associated disability, dementia and ultimately death worldwide. Although oxidative stress has been intensely studied, little analysis has been done in chronic oxidative stress-induced mitochondrial models. In this regard, DNA-overproliferation and/or deletion initiate mitochondrial deregulation causing energy failure, which has been implicated in the pathogenesis of Alzheimer’s disease (AD), tumor growth, and metastasis. In this regard, decline in mitochondrial normal homeostasis during the development and maturation of the neurodegeneration, tumor growth, and/or metastases is characterized by tissue and cellular oxygen deficiency, which leads to subcellular energy defects. In addition, the overexpression of the cascades initiates the formation and release of large amounts of reactive free radicals [mainly nitric oxide (NO) via the overexpression of NO synthases], which cause the oxidative stress, cellular alterations, and concomitant mitochondrial lesions and decline in normal organ function. The present study explores the intimate, i.e. direct relationship between chronic oxidative stress and mitochondrial damage as a vital life-supporter for cells and/or the microcirculatory systems whose damage occurs before the development of human AD. Our study highlights the effects of chronic oxidative stress-induced mitochondrial DNA over-proliferation and/or deletion and mitochondrial enzyme activities during the development of human AD. Mitochondrial DNA damage also leads to other pathologies, including colorectal cancer in liver metastasis, and malignant brain cancers. We hypothesize that mitochondrial lesions, especially mitochondrial DNA abnormalities, are detrimental to cell viability and thus mitochondrial DNA damage could be used as a new diagnostic tool and/or criterion for the early detection of AD and other diseases. Further extension of this approach will enable us not only for the better understanding of the blood brain barrier (BBB) homeostasis, which most likely plays a key role in the development of AD and some of forms of the cancer, but also for the development of new and more specific treatment strategies.

&amp;lt;i&amp;gt;Helicobacter pylori&amp;lt;/i&amp;gt;Induction in Gastric Mucosal Prostaglandin and Nitric Oxide Generation Is Dependent on MAPK/ERK-Mediated Activation of IKK-&amp;lt;i&amp;gt;β&amp;lt;/i&amp;gt; and cPLA&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;: Modulatory Effect of Ghrelin

Among the key factors defining the extent of gastric mucosal inflammatory involvement in response to H. pylori is the excessive generation of prostaglandin (PGE2) and nitric oxide (NO), caused by the overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and triggered by the activation of MAPK/JNK, p38 and ERK, and nuclear translocation of the cognate transcription factors. In this study, we report on the role of MAPK/ERK in the regulation of H. pylori LPS-induced gastric mucosal expression of COX-2 and iNOS. We show that ERK activation by the LPS leads to phosphorylation of the inhibitory κB kinase-β (IKK-β) and cytosolic phospholipase A2 (cPLA2), and is reflected in the upsurge in NF-κB nuclear translocation, induction in COX-2 and iNOS expression, and up-regulation in cPLA2 activity. The modulatory effect of peptide hormone, ghrelin, on the LPS-induced changes, although associated with further enhancement in ERK, IKK-β and cPLA2 phosphorylation, was reflected in the suppression of IKK-β and cPLA2 activity through S-nitrosylation. While the effect of ghrelin on S-nitrosylation was susceptible to suppression by the inhibitors of Src/Akt pathway, the inhibition of ERK activation caused the blockage in IKK-β and cPLA2 phosphorylation as well as S-nitrosylation. Taken together, our data show that H. pylori-induced ERK activation plays a critical role in up-regulation of gastric mucosal PGE2 and NO generation at the level of IKK-β and cPLA2 activation, and that ghrelin counters these proinflammatory consequences of the LPS through Src/Akt-dependent S-nitrosylation.