Presence Of iNOS Research Articles

Inducible nitric oxide synthase controls experimental Strongyloides infection.

Infection with Strongyloides sp. induces a host immune response, predominantly the Th2 type, that is able to eliminate the parasite. However, little is known about the role of the nitric oxide (NO) mediator, induced by the enzyme nitric oxide synthase (NOS), in strongyloidiasis. Therefore, in this study, we investigated the immune response of mice genetically deficient in the enzyme inducible nitric oxide synthase (iNOS-/- ), infected with Strongyloides venezuelensis. C57BL/6 wild-type (WT) and iNOS-/- mice were individually inoculated by subcutaneous injection of 3000 S.venezuelensis L3 larvae. In the absence of iNOS, mice were more susceptible to the infection than WT animals, in which the parasite was completely eliminated. The overall production of cytokines and specific IgG, IgG1 or IgE antibodies against the parasite was significantly lowered in infected iNOS-/- mice. The expression of iNOS was observed in the intestine of WT hosts but mainly in the wall of the parasite, despite the presence of iNOS in mice. Altogether, we concluded that iNOS expression may play an important role in the control of S.venezuelensis infection.

Mesenchymal stem cell secretome reduces pain and subchondral bone alterations in a mouse osteoarthritis model

Purpose: Mesenchymal stem cells (MSCs) represent a promising biological therapeutic option as an OA modifying treatment and have shown clinical improvement. Next to their differentiation potential into several lineages, they have the ability to influence their environment by secreting trophic mediators. These secreted factors could possibly counteract inflammatory and catabolic processes and attract endogenous repair cells. By using the MSC secretome as an OA treatment instead of the MSCs themselves, the possibilities to provide a more standardized and affordable therapeutic option would be largely increased. Together with a decrease of legal and safety issues, this would greatly enhance the clinical applicability of cell-based treatment as a true disease modifying anti-osteoarthritic drug. Aim of our study was to explore the anti-osteoarthritic effects of MSC secretome on pain, subchondral bone changes, cartilage damage, synovial thickness and synovial macrophage subtypes in a mouse osteoarthritis model. Methods: For the production of MSC Conditioned Medium (MSC-CM), human bone marrow derived MSCs from 3 donors were stimulated with IFNγ and TNFα (50 ng/mL each). After 24 hours of stimulation, medium was replaced by αMem without Phenol Red containing 0.05% BSA. This medium was collected after 24 hours, concentrated using 3k cut-off filters, frozen and designated MSC-CM. OA was induced unilaterally in 54 male C57/Bl6 mice by two intra-articular collagenase injections (collagenase-induced osteoarthritis model). After 7 days, mice were randomly assigned to 3 consecutive injections with 20.000 MSCs, MSC-CM from 20.000 MSCs or medium only (control). Weight distribution over the left and right hind limbs was evaluated as an indicator of pain using an incapitance tester. Other outcome measures were evaluated by histology at 5 and 21 days after treatment. Results: Seven days after induction of OA (day 0), 41.2 6.3% of weight was distributed on the affected limb, indicating pain when compared to before OA (Fig. 1, P<0.0001). We observed an early normalization in weight bearing in MSC-CM injected animals at day 7 compared to day 0, indicating early pain reduction (P = 0.001). Moreover, mice that received MSC-CM displayed significantly less pain compared to the medium control at day 7 (P = 0.001). OA induction lead to loss of subchondral bone tissue in the medial tibia plateau, 5 days after the first collagenase injection. (Fig. 2, P = 0.003). The percentage of subchondral bone in the MSC-CM treated group as well as the MSC treated group was not statistically different from the healthy control group, indicating amelioration of OA related decrease of subchondral bone volume. No treatment effects were seen on cartilage damage, synovial inflammation or the presence of iNOS, CD206, or CD163 positive macrophages (Fig. 3A–C). Conclusions: In this study, we found that injection of human MSC secretome resulted in an early pain reduction and amelioration of early subchondral bone changes, in a comparable manner to injection of MSCs themselves. Possibly, these two aspects are related, since subchondral bone marrow edema or bone marrow lesions have clinically been shown to be correlated with patients perceived degree of pain. Synovitis is another OA feature which has clinically been related to pain. We did not find a treatment effect on synovial thickness or macrophage phenotype in our study. Additional studies are warranted to further unravel the mechanisms behind the therapeutic effects of MSCs and their secretome. This way, we can evolve this biological approach towards a true disease modifying anti-osteoarthritic drug, in which the use of secretome would greatly enhance the standardization, affordability and clinical translatability.

Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells

Background/Aims: This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). Methods: We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO₃) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3’,5’ monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. Results: Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. Conclusion: These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.

The role of inducible nitric oxide synthase in urinary bladders of cattle with enzootic hematuria and its struggle with uroplakin III

Changes in uroplakin secretion in the urothelium leads a gradual ability lost in neoplastic conditions. Urothelium is one of the target regions where nitric oxide can damage. Therefore, in this study, the presence of iNOS and UPIII were investigated in bovine enzootic hematuria cases. The results were supported by comparing them with those achieved by critical markers for malignancy of tumor such as caspase 3, Bax, p53, PCNA, and VEGF. The presence of 8-OHDG, a good marker for oxidative DNA damage, was also investigated. For this purpose, urinary bladders with enzootic hematuria were collected from the slaughterhouses. In histopathological examinations, neoplastic, pre-neoplastic, and inflammatory lesions were observed in these urinary bladders. In immunohistochemical examinations, iNOS reacts as negative in the UPIII positive healthy urothelium which preserves the structure of AUM. On the other hand positive reaction has been observed in AUM modified and UPIII negative urothelium. In these cases, as an indicator of oxidative damage, 8-OHdG positivity was observed in the UPIII negative and iNOS positive areas. Additionally, increased cell proliferation, decreased apoptosis as well as p53 disorganization were clearly observed in most of the cases. Thus, the active role of nitric oxide, as a free radical derivative, has been shown in the enzootic hematuria cases of cattle.

The role of iNOS in cholesterol-induced liver fibrosis

Accumulation of cholesterol in the liver is associated with the development of non-alcoholic steatohepatitis-related fibrosis. However, underlying mechanisms are not well understood. The present study investigated the role of inducible nitric oxide synthase (iNOS) in cholesterol-induced liver fibrosis by feeding wild-type (WT) and iNOS-deficient mice with control or high-cholesterol diet (HCD) for 6 weeks. WT mice fed with HCD developed greater liver fibrosis, compared with iNOS-deficient mice, as evident by Sirius red staining and higher expression levels of profibrotic genes. Enhanced liver fibrosis in the presence of iNOS was associated with hypoxia-inducible factor-1α stabilization, matrix metalloproteinase-9 expression, and enhanced hepatic DNA damage. The profibrotic role of iNOS was also demonstrated in vivo using a selective inhibitor of iNOS as well as in vitro in a rat liver stellate cell line (HSC-T6). In conclusion, these findings suggest that iNOS is an important mediator in HCD-induced liver fibrosis.

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.

Interaction of Inducible Nitric Oxide Synthase with Rac2 Regulates Reactive Oxygen and Nitrogen Species Generation in the Human Neutrophil Phagosomes: Implication in Microbial Killing

Present study explores importance of inducible nitric oxide synthase (iNOS) and its interaction with Rac2 in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, protein-nitration and in microbial killing by neutrophils. The iNOS transcript and protein were constitutively present in human as well as in mice neutrophils. iNOS protein was found in cytosol, granules containing elastase and gelatinase, and in other subcellular organelles in resting human neutrophils. After phagocytosis of bovine serum albumin (BSA) coated beads, both human and mice neutrophils showed significant elevation in superoxide radicals, nitric oxide (NO), ROS/RNS and consequent BSA nitration. These responses were significantly reduced in presence of iNOS, NADPH oxidase (NOX), myeloperoxidase or Rac inhibitors, as well as in iNOS, Nox2 and Rac2 silenced human or iNOS-knockout mice neutrophils. Complex formed on interaction of iNOS with Rac2 coprecipitated with anti-Rac2, predominantly in cytosol in resting human neutrophils, while iNOS-Rac2 complex translocated to phagosomes after phagocytosis. This was accompanied by generation of superoxide radicals, NO, ROS/RNS and consequent BSA-nitration. Importance of Rac2 in iNOS mediated NO formation and microbial killing was confirmed by pretreatment of mice with Rac inhibitor, NSC23766 that significantly abrogated NO release and microbial killing in vivo. Present study highlights previously undefined role of Rac2-iNOS interaction, in translocation of iNOS to phagosomal compartment and consequent NO, superoxide radicals, ROS/RNS generation, BSA nitration and microbial killing. Altogether results obtained demonstrate the role of iNOS in NO and ROS/RNS generation, after phagocytosis of coated latex beads by human polymorphonuclear neutrophils. These studies imply functional importance of iNOS and its interaction with Rac2 in pathogen killing by the neutrophils.

Phenotype of Transgenic Mice Overexpressed with Inducible Nitric Oxide Synthase in the Retina

BackgroundUnlike its constitutive isoforms, including neuronal and endothelial nitric oxide synthase, inducible nitric oxide synthase (iNOS) along with a series of cytokines are generated in inflammatory pathologic conditions in retinal photoreceptors. In this study, we constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage.MethodsFor construction of opsin/iNOS transgene in the CMVSport 6 expression vector, the 4.4 kb Acc65I/Xhol mouse rod opsin promoter was ligated upstream to a 4.1 kb fragment encoding the complete mouse cDNA of iNOS. From the four founders identified, two heterozygote lines and one homozygote line were established. The presence of iNOS in the retina was confirmed and the pathologic role of iNOS was assessed by detecting nitrotyrosine products and apoptosis. Commercial TUNEL kit was used to detect DNA strand breaks, a later step in a sequence of morphologic changes of apoptosis process.ResultsThe insertion and translation of iNOS gene were demonstrated by an intense single 130 kDa band in Western blot and specific immunolocalization at the photoreceptors of the retina. Cellular toxicity in the retinas of transgenic animals was detected by a post-translational modification product, tyrosine-nitrated protein, the most significant one of which was nitrated cytochrome c. Following the accumulation of nitrated mitochondrial proteins and cytochrome c release, marked apoptosis was detected in the photoreceptor cell nuclei of the retina.ConclusionsWe have generated a pathologic phenotype with sustained iNOS overexpression and, therefore, high output of nitric oxide. Under basal conditions, such overexpression of iNOS causes marked mitochondrial cytochrome c nitration and release and subsequent photoreceptor apoptosis in the retina. Therefore, the modulation of pathways leading to iNOS generation or its effective neutralization can be of significant therapeutic benefit in the oxidative stress-mediated retinal degeneration, a leading cause of blindness.

Abstract 461: Involvement of α9β1 integrin-iNOS pathway in matrix metalloproteinase-9 and/or urokinase plasminogen activator receptor-mediated glioma cell migration

Abstract Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide (NO) production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we aimed to investigate the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Immunohistochemical analysis of the glioblastoma multiforme (GBM) tissue array revealed the prominent presence of iNOS in several clinical GBM samples. In addition, immunofluorescence analysis of U251 glioma cell lines and 5310 glioma xenograft cells showed a prominent expression of iNOS. Transcriptional inactivation of MMP-9 and/or uPAR by respective shRNA reduced iNOS expression in both U521 and 5310 glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in these glioma cells after transfection with MMP-9- or uPAR-overexpressing plasmids. Wound healing and spheroid migration assays showed a significant inhibition of the migration potential of MMP-9- or uPAR-overexpressed U251 glioma cells after treatment with L-NAME, an inhibitor of iNOS. Similarly, Matrigel invasion assay revealed a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed U251 and 5310 glioma cells after L-NAME treatment. Further, we noticed a prominent reduction of iNOS expression in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Integrin α9β1 regulates iNOS activity via Src tyrosine kinase; Src coordinates subsequent signaling pathways through activation of FAK and tyrosine phosphorylation of the adaptor protein p130Cas. Western blot analysis of MMP-9 and/or uPAR shRNA treated U251 and 5310 glioma cells revealed significant reductions in the protein expression of cSrc, phosphoSrc, FAK and p130Cas after simultaneous downregulation of both MMP-9 and uPAR. Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of these glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels. Therefore, our results highlight the possible involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 461. doi:1538-7445.AM2012-461

Renal actions of atrial natriuretic peptide in spontaneously hypertensive rats: the role of nitric oxide as a key mediator

Atrial natriuretic peptide (ANP) is an important regulator of blood pressure (BP). One of the mechanisms whereby ANP impacts BP is by stimulation of nitric oxide (NO) production in different tissues involved in BP control. We hypothesized that ANP-stimulated NO is impaired in the kidneys of spontaneously hypertensive rats (SHR) and this contributes to the development and/or maintenance of high levels of BP. We investigated the effects of ANP on the NO system in SHR, studying the changes in renal nitric oxide synthase (NOS) activity and expression in response to peptide infusion, the signaling pathways implicated in the signaling cascade that activates NOS, and identifying the natriuretic peptide receptors (NPR), guanylyl cyclase receptors (NPR-A and NPR-B) and/or NPR-C, and NOS isoforms involved. In vivo, SHR and Wistar-Kyoto rats (WKY) were infused with saline (0.05 ml/min) or ANP (0.2 μg·kg(-1)·min(-1)). NOS activity and endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) NOS expression were measured in the renal cortex and medulla. In vitro, ANP-induced renal NOS activity was determined in the presence of iNOS and nNOS inhibitors, NPR-A/B blockers, guanine nucleotide-regulatory (G(i)) protein, and calmodulin inhibitors. Renal NOS activity was higher in SHR than in WKY. ANP increased NOS activity, but activation was lower in SHR than in WKY. ANP had no effect on expression of NOS isoforms. ANP-induced NOS activity was not modified by iNOS and nNOS inhibitors. NPR-A/B blockade blunted NOS stimulation via ANP in kidney. The renal NOS response to ANP was reduced by G(i) protein and calmodulin inhibitors. We conclude that ANP interacts with NPR-C, activating Ca-calmodulin eNOS through G(i) protein. NOS activation also involves NPR-A/B. The NOS response to ANP was diminished in kidneys of SHR. The impaired NO system response to ANP in SHR participates in the maintenance of high blood pressure.

High iNOS mRNA and protein localization during late pregnancy suggest a role for nitric oxide in mouse pubic symphysis relaxation

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone-controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast-like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on Day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL.

Prediction of nitric oxide concentrations in melanomas

The presence of iNOS and nitrotyrosine in cutaneous melanomas has been correlated with poor survival rates of patients, suggesting that NO plays a role in the tumor pathophysiology. However, the concentrations of NO that melanoma cells are exposed to in vivo have been unknown. To provide cell kinetic data for use in predicting those concentrations, synthesis and consumption of NO was examined in A375 melanoma cells. Nitric oxide synthesis was undetectable. The rate of intracellular NO consumption was determined by continuous monitoring of NO concentrations following injection of NO solutions in a closed chamber. After correcting for autoxidation and consumption from media-generated O 2 - , the rate constant obtained for cellular consumption was 7.1 ± 1.1 s −1. This information was combined with previous data on macrophage NO kinetics to develop a mathematical model to predict NO levels in cutaneous melanomas. Synthesis of NO by macrophages in the stroma was found to give a maximum concentration at the tumor periphery of 0.2 μM. Because of the high rates of cellular consumption, the elevation in NO concentration is predicted to be very localized, approximately 90% of the concentration decay occurring within 30 μm of the tumor edge. High NO concentrations at the periphery of a melanoma may contribute to metastasis by stimulating cell proliferation, inhibiting apoptosis, or acting as a lymphangiogenic factor.

Effect of Hypertonic Saline Resuscitation on CD4+CD25+ Regulatory T Cells and γδ T Cells After Hemorrhagic Shock and Resuscitation in Relation to Apoptosis and iNOS

Hemorrhagic shock and resuscitation induce immunosuppression. CD4CD25 regulatory T cells and gammadeltaT cells may affect these immunosuppressive conditions. Hypertonic saline resuscitation reduces damage to organs and apoptosis and also restores immunosuppressive condition. We investigated how hypertonic saline resuscitation affected the induction of CD4CD25 regulatory T cells and gammadeltaT cells, and their apoptosis after hemorrhagic shock and resuscitation, and its relationship to inducible nitric oxide synthase (iNOS) (nitric oxide production). Male inbred C57BL6/J mice 8-week to 12-week-old as wild type and iNOS gene knock out (iNOS-/-), weighing 20 g to 35 g, were used. Hemorrhagic shock model of +/-40 mm Hg for 60 minutes was setup. Animals were randomly assigned to the following four resuscitation group: (1) wild HS: resuscitation with hypertonic saline (4 mL/Kg of 7.5% NaCl) and shed blood (SB), (2) wild 2LR: resuscitation with lactated Ringer's solution (two times the volume of the SB) and SB, (3) iNOS knockout HS, and (4) iNOS knockout 2LR. Untreated groups for wild and iNOS knockout mice were designated as control groups. Samples of thymus and spleen were harvested at 2 hours, 6 hours, 24 hours, and 48 hours after resuscitation. CD4CD25 regulatory T cells and gammadeltaT cells were analyzed using three-color flow cytometry. (1) gammadelta T cells increased earlier at 24 hours and CD4CD25 regulatory T cells increased later at 48 hours compared with controls in spleen of wild type (p < 0.01). (2) Hypertonic saline resuscitation suppressed gammadelta T cells compared with 2LR at 24 hours in iNOS knockout mice in spleen (p < 0.05). Hypertonic saline resuscitation increased apoptosis of CD4CD25 regulatory T cells at 48 hours in iNOS knockout mice in spleen (p < 0.01). (3) CD4CD25 regulatory T cells of iNOS knockout both in HS and 2LR groups at 48 hours decreased compared with wild type both in HS and 2LR groups in spleen (p < 0.01). (4) Apoptotic gammadelta T cells both in spleen and thymus in iNOS knockout mice at 48 hours increased compared with those in wild type (p < 0.05, respectively, except gammadelta T cells 2LR in spleen: p = 0.058). gammadelta T cells increased earlier at 24 hours, whereas CD4CD25 regulatory T cells increased later at 48 hours in spleen of wild type. Hypertonic saline was effective without the presence of iNOS, i.e., decreased gammadelta T cells at 24 hours and increased apoptosis of CD4CD25 regulatory T cells at 48 hours. CD4CD25 regulatory T cells at 48 hours without iNOS decreased compared with those of wild type. gammadelta T cells at 48 hours induced apoptosis under the condition without iNOS in spleen and thymus. iNOS worked as an accelerating factor for immunosuppressive condition, affected apoptosis, and immunoenhancing effect by hypertonic saline.

Role of nitric oxide as a key mediator on cardiovascular actions of atrial natriuretic peptide in spontaneously hypertensive rats

The objective was to study atrial natriuretic peptide (ANP) effects on mean arterial pressure (MAP) and cardiovascular nitric oxide (NO) system in spontaneously hypertensive rats (SHRs), investigating the receptors and signaling pathways involved. In vivo, SHRs and Wistar-Kyoto (WKY) rats were infused with saline (0.05 ml/min) or ANP (0.2 microg.kg(-1).min(-1)) for 1 h. MAP and nitrites and nitrates excretion (NOx) were determined. NO synthase (NOS) activity and endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) NOS expression were measured in the heart and aorta. In vitro, heart and aortic NOS activity induced by ANP was determined in the presence of iNOS and nNOS inhibitors, natriuretic peptide receptor (NPR)-A/B blocker, G(i) protein, and calmodulin inhibitors. As a result, ANP diminished MAP and increased NOx in both groups. Cardiovascular NOS activity was higher in SHRs than in WKY rats. ANP increased NOS activity, but the activation was lower in SHRs than in WKY rats. ANP had no effect on NOS isoform expression. NOS activity induced by ANP was not modified by iNOS and nNOS inhibitors. NPR-A/B blockade blunted NOS stimulation via ANP in ventricle and aorta but not in atria. Cardiovascular NOS response to ANP was reduced by G(i) protein and calmodulin inhibitors in both groups. In conclusion, in atria, ventricle, and aorta, ANP interacts with NPR-C receptors, activating Ca(2+)-calmodulin eNOS through G(i) protein. In ventricle and aorta, NOS activation also involves NPR-A/B. The NOS response to ANP was impaired in heart and aorta of SHRs. The impaired NO-system response to ANP in hypertensive animals, involving alterations in the signaling pathway, could participate in the maintenance of high blood pressure in this model of hypertension.

Alveolar macrophage inducible nitric oxide synthase-dependent pulmonary microvascular endothelial cell septic barrier dysfunction

Inducible nitric oxide (NO) synthase (iNOS) from neutrophils and alveolar macrophages (AM) contributes to the pathophysiology of murine septic acute lung injury (ALI). It is not known if AM iNOS has a direct effect on septic pulmonary microvascular endothelial cell (PMVEC) permeability. We hypothesized that AM iNOS mediates PMVEC permeability in vitro under septic conditions through NO and peroxynitrite. 100,000 confluent PMVEC on cell-culture inserts were co-incubated with iNOS+/+ vs. iNOS−/− AM, in various ratios of AM to PMVEC. PMVEC injury was assessed by trans-PMVEC Evans Blue-labelled albumin flux in the presence or absence of cytomix (equimolar TNF-α, IL-1β and IFN-γ). Cytomix stimulation dose-dependently increased trans-PMVEC EB-albumin flux, which was exaggerated (1.4±0.1% vs. 0.4±0.1% in unstimulated PMVEC, p<0.05) in the presence of iNOS+/+, but not iNOS−/−, AM in the upper compartment. Similarly, iNOS+/+, but not iNOS−/−, AM in the lower compartment also enhanced septic trans-PMVEC albumin leak. The mechanism of iNOS-dependent septic PMVEC permeability was pursued through pharmacologic studies with inhibitors of NOS, and scavengers of NO, superoxide, and peroxynitrite, and treatment of PMVEC with the NO donor, DETA-NONOate. Septic iNOS+/+ AM-dependent trans-PMVEC albumin leak was significantly attenuated by pharmacologic iNOS inhibition (L-NAME and 1400W), and scavenging of either NO (oxyhemoglobin), superoxide (PEG-SOD), or peroxynitrite (FeTPPS). Exogenous NO (DETA-NONOate) had no effect on PMVEC permeability. These data are consistent with a direct role of AM iNOS in septic PMVEC barrier dysfunction, which is likely mediated, in part, through peroxynitrite.

Lymphotoxin is critically required for the presence of IgA‐promoting iNOS+ DCs in the intestinal lamina propria

Lymphotoxin‐β receptor (LTβR) signaling is essential for IgA production in the intestinal lamina propria (LP) and it has been proposed that LTβR‐signaling is needed for the accumulation of IgA‐secreting B‐1 cells in the LP. Yet, in spite of diminished numbers of B‐1 cells in the LP of LTβR−/− mice, a residual population of B‐1 cells remain and B‐2 cell numbers in the LP are normal. To further explore the mechanism for LTβR‐dependent IgA production in the gut, we hypothesized that LTβR signaling may regulate accessory cells in the LP that influence IgA production by LP‐resident B cells. The LP harbors a recently described subset of iNOS+ dendritic cells (DC) that are required for IgA responses. We characterized this DC population in the LP of WT mice as CD11c+ CD11blo Thy1+ CD8α − B220− and, interestingly, we found that these cells were essentially undetectable in LTβR−/ − mice. In mixed bone marrow chimeras, the presence of iNOS+ DC in the LP depended primarily on the expression of LTβR by radio‐resistant cells, whereas the expression of LTβR on DC themselves was not essential. We conclude that the presence of IgA‐promoting iNOS+ DCs in the LP requires intact LTβR signaling on the LP stroma, and these data provide a mechanism for defective IgA production in LTβR−/ − mice. This work was supported by the Canadian Institutes of Health Research.

Two Autocrine Pathways to Regulate Cyclic GMP Synthesis in Cultured Human Retinal Pigment Epithelial Cells

Aim: To investigate the role of two separate enzymatic pathways [soluble (sGC) vs. particulate (pGC) guanylyl cyclase] in the synthesis of cyclic GMP (cGMP) in cultured human retinal pigment epithelial (RPE) cells. Methods: cGMP accumulation was evaluated by quantitative analysis of cGMP immunoreactivity. RPE cells were also stained for inducible nitric oxide synthase (iNOS), ANP and β₁- and α₂-subunits of sGC. Results: We showed nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and iNOS immunoreactivity in RPE cells. Incubation of the cells in the presence of 1 mM IBMX to inhibit phosphodiesterase activity, and the simultaneous inhibition of NOS activity with L-NAME suggested the involvement of sGC in maintaining a low level of cGMP in the RPE cells. The involvement of sGC was further supported by detection of the β₁- and α₂-subunits of sGC. Incubation of the cells in the presence of atrial natriuretic peptide (ANP) to stimulate pGC strongly increased cGMP immunoreactivity. We also demonstrated the presence of ANP in all RPE cells. Conclusion: Cultured human RPE cells are capable of producing cGMP after stimulation of sGC or pGC. The presence of iNOS and ANP in all cells suggests two different autocrine pathways of stimulating cGMP production in these cells. The possible role of cGMP in the regulation iNOS gene expression and in the regulation of ANP is discussed.

Different expression of NOS isoforms in early endothelial progenitor cells derived from peripheral and cord blood

Cord blood and peripheral-adult blood were compared as different sources of early endothelial precursor cells (eEPCs). Total mononuclear cells (MNCs) were obtained from both blood types and committed to eEPCs by exposure to fibronectin, VEGF, IGF-I, and bFGF. Under this condition, MNCs seeded at the density of 3 x 10(5) cells/cm(2) assumed a spindle shape, which was indicative of developing eEPCs, and expanded in a similar manner irrespective to the blood sources. Ulex europaeus agglutinin (UEA-1) and acetylated low density lipoprotein (acLDL) double staining was present in 90% in both peripheral- and cord-blood eEPCs after 2-week expansion. Also, the ability of eEPCs to form tubule-like structures in Matrigel was independent of their blood source, but dependent on the presence of human umbilical vein endothelial cells (HUVECs). eNOS and nNOS were not detectable by Western blotting in both peripheral and cord-blood eEPCs upon 3 weeks and their mRNA levels were lower than 2% relative to those present in HUVECs. On the contrary, iNOS protein was detectable in peripheral-blood eEPCs, but not in cord-blood eEPCs and HUVECs, as well as iNOS mRNA was more concentrated in peripheral-blood eEPCs than in cord-blood eEPCs and HUVECs. These data suggest that: (a) peripheral and cord blood can be considered comparable sources of eEPCs when they are expanded and differentiated in a short-term period; (b) the extremely low expression of constitutive NOS isoforms in the eEPCs of both blood types should markedly reduce their ability to regulate NO-dependent vasorelaxation; (c) the presence of iNOS in peripheral-blood eEPCs could improve the process of vasculogenesis.

NO and Central Cardiovascular Control

The 1998 Nobel Prize in Physiology or Medicine recognized the great contributions of Robert Furchgott, Louis Ignarro, and Ferid Murad through “their discoveries concerning nitric oxide as a signaling molecule in the cardiovascular system.” Furchgott’s earlier work describing a vasodilatory factor that was activated by acetylcholine acting on an intact endothelium not only set the stage for subsequent recognition that NO was the endothelium-derived relaxation factor but also acted as a harbinger of much subsequent study of interactions between classic neurotransmitters and NO. Three isoforms of NO synthase (NOS), the biosynthetic enzyme for synthesis of NO, have been identified. Two are constitutive enzymes, endothelial NOS (eNOS) and neuronal NO (nNOS). Each has been identified in the nucleus tractus solitarii (NTS), the primary site of termination of baroreceptor afferent nerves, but the role of each, or for that matter the role of NO itself, in baroreflex transmission through the NTS remains very much in question. The presence of iNOS in the NTS could indicate that there had been stress in animals in which it was found. In keeping with the potential that NO may link transmitter mechanisms and may integrate vascular and neuronal functions, the work by Waki et al in this issue of Hypertension 1 provides further evidence for complex integration through NO mechanisms in the NTS and for contributions of NO in disorders of cardiovascular homeostasis. That work is the latest from the Paton/Kasparov laboratories at the University of Bristol and further supports there being a cascade of transduction events that involve NO derived from eNOS, as well as several additional putative transmitters in NTS regulation of blood pressure. One such potential transmitter is angiotensin. Particularly since the work of Casto and Phillips,2 it has been recognized that angiotensin may act at the level of the NTS to …

Tumor iNOS predicts poor survival for stage III melanoma patients

Inducible nitric oxide synthase (iNOS) produces nitric oxide, which has growth promoting activity in melanoma. A preliminary study of tumors from patients with Stage III melanoma who had received neo-adjuvant therapy revealed an association of tumor iNOS expression with shortened survival. The objective of the present study was to determine whether iNOS expression in tumors of newly diagnosed, untreated Stage III patients is predictive of survival. iNOS expression was examined by immunohistochemistry in tumors from 132 patients. The staining was evaluated for percentage of positive cells (Number score) and the intensity of staining (Intensity score). The association of iNOS expression with overall and disease-specific survival was tested in univariate and multivariate Cox proportional hazards regression models that included other known prognostic factors. Results of the univariate analysis demonstrated that the presence of iNOS in a patient's tumor, whether graded on the basis of Number or Intensity score, was associated with a significant increase in the hazard ratio of death from melanoma. These findings were corroborated by median survival data estimated from Kaplan Meier analysis. In the multivariate model including iNOS number or intensity, gender, age, number of lymph nodes, macroscopic disease and in-transit disease, only iNOS expression predicted survival. We conclude that a significant association exists between tumor iNOS expression and shortened survival in untreated Stage III melanoma patients. The ability of iNOS to predict outcomes for these patients may be independent of other known prognostic factors, providing a new molecular marker with significant potential for clinical utility.