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International audience; BACKGROUND & AIMS: Liver resection includes temporal vascular inflow occlusion resulting in ischemia/reperfusion injury in the remnant liver. Here, we developed a rat model of selective lobe occlusion to isolate reperfusion stress from ischemia and to analyze its effect on liver regeneration. METHODS: Left lateral and median lobes of liver were either mobilized or subjected twice for 10min to ischemia followed by 5min reperfusion prior to resection while the regenerative lobes were only subjected to reperfusion. RESULTS: Although intermittent reperfusion stress induced higher levels of serum transaminases, analysis of cell cycle regulators revealed accelerated regenerative response compared to standard partial hepatectomy. The G0/G1 transition occurred before tissue resection, as evidenced by c-fos, junB, and IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition, and cell division occurred earlier than normal. Unexpectedly, liver mobilization, a component of the clamping procedure, also resulted in earlier G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal Kinase pathway and was associated with an oxidative stress response as evidenced by the expression of inducible nitric oxide synthase. CONCLUSION: Intermittent selective clamping of lobes to be resected induced reperfusion stress on remnant liver that was beneficial for liver regeneration, suggesting this procedure could be applied in clinical practice.

Toll-like receptor 2 (TLR2) signaling has been shown to contribute to resistance to Listeria monocytogenes infection, as TLR2-deficient mice have a heightened susceptibility to infection with this organism. Because CD14 may associate with TLR2, we investigated the role of CD14 in Listeria responses. In both CD14-deficient and TLR2-deficient macrophages, nuclear factor kappaB translocation; CD40 and CD86; and the production of interleukin (IL)-12, IL-6, tumor necrosis factor, and nitric oxide are reduced. The absence of CD14 augmented susceptibility to Listeria infection, reduced survival, and diminished bacterial clearance, as observed in TLR2-deficient mice. Compared with C57BL/6 control mice, CD14-deficient mice were observed to have a greater number of hepatic microabscesses containing abundant neutrophils, these abscesses were larger in size, and there was reduced inducible nitric oxide synthase expression. Further, mice that are both CD14 deficient and TLR2 deficient display susceptibility to infection that is comparable to that of mice deficient in either CD14 or TLR2 alone. Therefore, the present data demonstrate the role of CD14 and TLR2 in the recognition and control of Listeria infection and host resistance.

Pathogens contain specific pathogen-associated molecular patterns, which activate pattern recognition receptors of the innate immune system such as Toll-like receptors (TLRs). Although there is a clear evidence of how macrophages sense pathogens, we know less about such processes in vessels. This is critical to understand because activation of vascular cells and the subsequent induction of inflammatory genes by bacteria are crucial events in the development of septic shock. In the current study we have used genetically modified mice to investigate the role of TLRs, adapter proteins, tumor necrosis factor alpha (TNFalpha), and nitric oxide synthase II (NOSII) in vascular dysfunction induced by Gram-positive (Staphylococcus aureus) or Gram-negative (Escherichia coli) bacteria. Our data show that Gram-positive S. aureus or Gram-negative E. coli causes vascular dysfunction via the induction of NOSII. For S. aureus, this process requires TLR2, TLR6, myeloid differentiation factor 88 (MyD88) adapter-like, MyD88, and TNF, but not TLR4 or TLR1. Vascular dysfunction induced by E. coli requires TLR4 but has no requirement for TLR2, TLR1, TLR6, or TNF, and a partial but incomplete requirement of MyD88 and TIR domain-containing adapter inducing interferon-beta. Staphylococcus aureus induced NOSII protein expression in vascular smooth muscle cells but not in macrophages, whereas E. coli induced NOSII in both cell types. Our data are the first to establish the definitive roles of specific TLRs in the sensing of Gram-positive and Gram-negative bacteria by vessels and demonstrate that macrophages and blood vessels may differ in their response to pathogens.

In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was recently postulated that in humans, Brucella infection tends to become chronic because NO cannot exert its deleterious effect. In fact, conditions allowing NO production by human macrophages in culture are poorly defined, rendering the in vitro study of NO function difficult. Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, we showed that the intracellular development of Brucella was impaired in human macrophages, which produced NO. Although Brucella-infected human macrophagic phagocytes did not release NO in commonly used models of infection, the machinery required to produce NO was expressed in these cells and could be triggered by cell membrane receptors present on the infected cells. Therefore, the lack of NO production in isolated human macrophages infected by Brucella under in vitro conditions did not exclude a possible involvement of NO in the control of human brucellosis.

International audience; Nitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form.

International audience; Inflammation-mediated neurodegeneration occurs in the acute and the chronic phases of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Classically activated (M1) microglia are key players mediating this process. Here, we identified Galectin-1 (Gal1), an endogenous glycan-binding protein, as a pivotal regulator of M1 microglial activation that targets the activation of p38MAPK-, CREB-, and NF-κB-dependent signaling pathways and hierarchically suppresses downstream proinflammatory mediators, such as iNOS, TNF, and CCL2. Gal1 bound to core 2 O-glycans on CD45, favoring retention of this glycoprotein on the microglial cell surface and augmenting its phosphatase activity and inhibitory function. Gal1 was highly expressed in the acute phase of EAE, and its targeted deletion resulted in pronounced inflammation-induced neurodegeneration. Adoptive transfer of Gal1-secreting astrocytes or administration of recombinant Gal1 suppressed EAE through mechanisms involving microglial deactivation. Thus, Gal1-glycan interactions are essential in tempering microglial activation, brain inflammation, and neurodegeneration, with critical therapeutic implications for MS.

International audience; Control of pulmonary pathogens constitutes a challenging task as successful immune responses need to be mounted without damaging the lung parenchyma. Using immunofluorescence microscopy and flow cytometry, we analyzed in the mouse the initial innate immune response that follows intranasal inoculation of Brucella abortus. Bacteria were absent from parenchymal dendritic cells (DC) but present in alveolar macrophages in which they replicated. When the number of alveolar macrophages was reduced prior to Brucella infection, small numbers of pulmonary DC were infected and a massive recruitment of TNF-α- and iNOS-producing DC ensued. Coincidentally, Brucella disseminated to the lung-draining mediastinal lymph nodes (LN) where they replicated in both migratory DC and migratory alveolar macrophages. Together, these results demonstrate that alveolar macrophages are critical regulators of the initial innate immune response against Brucella within the lungs and show that pulmonary DC and alveolar macrophages play rather distinct roles in the control of microbial burden.

International audience; Alcohol consumption increases reactive oxygen species (ROS) formation, which can damage mitochondrial DNA (mtDNA) and alter mitochondrial function. To test whether manganese superoxide dismutase (MnSOD) modulates acute alcohol-induced mitochondrial alterations, transgenic MnSOD-overexpressing (MnSOD(+++)) mice, heterozygous knockout (MnSOD(+/-)) mice, and wild-type (WT) littermates were sacrificed 2 or 24 h after intragastric ethanol administration (5 g/kg). Alcohol administration further increased MnSOD activity in MnSOD(+++) mice, but further decreased it in MnSOD(+/-) mice. In WT mice, alcohol administration transiently increased mitochondrial ROS formation, decreased mitochondrial glutathione, depleted and damaged mtDNA, and decreased complex I and V activities; alcohol durably increased inducible nitric-oxide synthase (NOS) expression, plasma nitrites/nitrates, and the nitration of tyrosine residues in complex V proteins. These effects were prevented in MnSOD(+++) mice and prolonged in MnSOD(+/-) mice. In alcoholized WT or MnSOD(+/-) mice, mtDNA depletion and the nitration of tyrosine residues in complex I and V proteins were prevented or attenuated by cotreatment with tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a superoxide scavenger; N(omega)-nitro-l-arginine methyl ester and N-[3-(aminomethyl)benzyl]acetamidine (1,400W), two NOS inhibitors; or uric acid, a peroxynitrite scavenger. In conclusion, MnSOD overexpression prevents, and MnSOD deficiency prolongs, mtDNA depletion after an acute alcohol binge in mice. The protective effects of MnSOD, tempol, NOS inhibitors, and uric acid point out a role of the superoxide anion reacting with NO to form mtDNA-damaging peroxynitrite.

The contribution of lymphotoxin (LT)alpha in the host immune response to virulent Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin infections was investigated. Despite their ability to induce Th1 cytokine, IFN-gamma, and IL-12 pulmonary response, "conventional" LTalpha(-/-) mice succumb rapidly to virulent M. tuberculosis aerosol infection, with uncontrolled bacilli growth, defective granuloma formation, necrosis, and reduced pulmonary inducible NO synthase expression, similar to TNF(-/-) mice. Contributions from developmental lymphoid abnormalities in LTalpha(-/-) mice were excluded because hematopoietic reconstitution with conventional LTalpha(-/-) bone marrow conferred enhanced susceptibility to wild-type mice, comparable to conventional LTalpha(-/-) control mice. However, conventional LTalpha(-/-) mice produced reduced levels of TNF after M. bovis bacillus Calmette-Guerin infection, and their lack of control of mycobacterial infection could be due to a defective contribution of either LTalpha or TNF, or both, to the host immune response. To address this point, the response of "neo-free" LTalpha(-/-) mice with unperturbed intrinsic TNF expression to M. tuberculosis infection was investigated in a direct comparative study with conventional LTalpha(-/-) mice. Strikingly, although conventional LTalpha(-/-) mice were highly sensitive, similar to TNF(-/-) mice, neo-free LTalpha(-/-) mice controlled acute M. tuberculosis infection essentially as wild-type mice. Pulmonary bacterial burden and inflammation was, however, slightly increased in neo-free LTalpha(-/-) mice 4-5 mo postinfection, but importantly, they did not succumb to infection. Our findings revise the notion that LTalpha might have a critical role in host defense to acute mycobacterial infection, independent of TNF, but suggest a contribution of LTalpha in the control of chronic M. tuberculosis infection.

International audience; BACKGROUND: The implication of nitric oxide (NO) in the development of human African trypanosomiasis (HAT) using an animal model, was examined. The manner by which the trypanocidal activity of NO is impaired in the periphery and in the brain of rats infected with Trypanosoma brucei brucei (T. b. brucei) was analyzed through: (i) the changes occurring in NO concentration in both peripheral (blood) and cerebral compartments; (ii) the activity of nNOS and iNOS enzymes; (iii) identification of the brain cell types in which the NO-pathways are particularly active during the time-course of the infection. METHODOLOGY/PRINCIPAL FINDINGS: NO concentration (direct measures by voltammetry) was determined in central (brain) and peripheral (blood) compartments in healthy and infected animals at various days post-infection: D5, D10, D16 and D22. Opposite changes were observed in the two compartments. NO production increased in the brain (hypothalamus) from D10 (+32%) to D16 (+71%), but decreased in the blood from D10 (-22%) to D16 (-46%) and D22 (-60%). In parallel with NO measures, cerebral iNOS activity increased and peaked significantly at D16 (up to +700%). However, nNOS activity did not vary. Immunohistochemical staining confirmed iNOS activation in several brain regions, particularly in the hypothalamus. In peritoneal macrophages, iNOS activity decreased from D10 (-83%) to D16 (-65%) and D22 (-74%) similarly to circulating NO. CONCLUSION/SIGNIFICANCE: The NO changes observed in our rat model were dependent on iNOS activity in both peripheral and central compartments. In the periphery, the NO production decrease may reflect an arginase-mediated synthesis of polyamines necessary to trypanosome growth. In the brain, the increased NO concentration may result from an enhanced activity of iNOS present in neurons and glial cells. It may be regarded as a marker of deleterious inflammatory reactions.

International audience; CX(3)CR1 expression is associated with the commitment of CSF-1R(+) myeloid precursors to the macrophage/dendritic cell (DC) lineage. However, the relationship of the CSF-1R(+) CX(3)CR1(+) macrophage/DC precursor (MDP) with other DC precursors and the role of CX(3)CR1 in macrophage and DC development remain unclear. We show that MDPs give rise to conventional DCs (cDCs), plasmacytoid DCs (PDCs), and monocytes, including Gr1(+) inflammatory monocytes that differentiate into TipDCs during infection. CX(3)CR1 deficiency selectively impairs the recruitment of blood Gr1(+) monocytes in the spleen after transfer and during acute Listeria monocytogenes infection but does not affect the development of monocytes, cDCs, and PDCs.

International audience; Glucagon-like peptide-1 (GLP-1) is a peptide released by the intestine and the brain. We previously demonstrated that brain GLP-1 increases glucose-dependent hyperinsulinemia and insulin resistance. These two features are major characteristics of the onset of type 2 diabetes. Therefore, we investigated whether blocking brain GLP-1 signaling would prevent high-fat diet (HFD)-induced diabetes in the mouse. Our data show that a 1-month chronic blockage of brain GLP-1 signaling by exendin-9 (Ex9), totally prevented hyperinsulinemia and insulin resistance in HFD mice. Furthermore, food intake was dramatically increased, but body weight gain was unchanged, showing that brain GLP-1 controlled energy expenditure. Thermogenesis, glucose utilization, oxygen consumption, carbon dioxide production, muscle glycolytic respiratory index, UCP2 expression in muscle, and basal ambulatory activity were all increased by the exendin-9 treatment. Thus, we have demonstrated that in response to a HFD, brain GLP-1 signaling induces hyperinsulinemia and insulin resistance and decreases energy expenditure by reducing metabolic thermogenesis and ambulatory activity.

Guest Editor; International audience; The study shows that the reduction in myocardial Cx43 content 2 weeks after cardiac infarction is less in iNOS−/− than in wild-type (WT) mice. Moreover, iNOS deficiency preserves the ratio of the phosphorylated to nonphosphorylated Cx43, improves cardiac function and increases survival. Finally, in vitro experiments demonstrate that exogenous NO production dose-dependently decreases Cx43 content in isolated cardiomyocytes.

International audience; Oxidative stress is a metabolic situation used by immune cells to provide protection against infection. Under activation by threatening elements, phagocytes produce chemically toxic molecules, namely the reactive oxygen species (ROS) and reactive nitrogen species (RNS). This mechanism involves two types of enzymes: NAPDH oxidases (NOX) and NO synthases (NOS), which activities are versatile and not fully understood yet. In this regard, we studied in real-time the release of bursts of ROS and RNS by single PLB-985 cells, originating from a myeloid cell line prone to differentiate into neutrophil or monocyte-like phagocytes. A selective electrochemical detection of each ROS or RNS was conducted at platinized carbon fiber microelectrodes positioned at micrometric distances from single cells. Our results show (1) the existence of a NO synthase activity in PLB-985 cells and (2) the ability of NO synthases to provide a NOX activity in cells where NADPH oxidase (NOX2) is knocked out.

International audience; In the present study, we have analyzed four highly polymorphic regions (STRs) chosen from four candidate genes involved in: (1) Platelet aggregation: alpha subunit of the platelet GpIIb/GpIIIa integrin complex (GpIIIa (CT)n; 17q21.31), (2) Coagulation fibrinolysis: Plasminogen Activator Tissue (PLAT5 (TG)14(TA)12; 8p12-q11.2) and Plasminogen Activator Inhibitor-1 (PAI-1 (CA)n; 7q21.3-q22), (3) Oxidative metabolism: the inducible nitric oxide (NO) synthase (iNOS) gene (NOS2A (CCTTT)n; 17cen-q11.2). Allele frequencies for these four STR loci were investigated in several Mediterranean populations. The population data deviate from the Hardy-Weinberg equilibrium in all populations for GpIIIa (CT)n polymorphism.

International audience; Myocardial infarction induces contractile dysfunction and remodeling that can lead to heart failure. Nitric oxide has been proposed as one of the major actors of this pathophysiologic process. We note that N (G)-nitro-L-arginine methyl ester (L-NAME) administration from day 2 to day 7 after myocardial infarction in rats improves stroke volume, preserves cardiac compliance, and reduces infarct expansion. Our observations lead to the hypothesis that the mechanisms by which cytokines contribute to myocardial remodeling and dysfunction in the days after infarction might involve *NO signalling pathways.

We show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Czeta (PKCzeta). To understand the effect of interleukin-13 or PKCzeta inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-alpha (TNF-alpha) with nuclear factor kappaB (NF-kappaB)/p65 expression. Treatment led to absence of NF-kappaB/p65 expression and low levels of TNF-alpha, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-kappaB/p65 decreased and nuclear NF-kappaB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-alpha, indicating onset of spontaneous resolution. Treatment limited PKCzeta cleavage, with expression of nuclear NF-kappaB/p50 and BCL-3 and low nuclear NF-kappaB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCzeta by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-alpha, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-kappaB/p65, increased transforming growth factor-beta2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3.

We show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Czeta (PKCzeta). To understand the effect of interleukin-13 or PKCzeta inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-alpha (TNF-alpha) with nuclear factor kappaB (NF-kappaB)/p65 expression. Treatment led to absence of NF-kappaB/p65 expression and low levels of TNF-alpha, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-kappaB/p65 decreased and nuclear NF-kappaB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-alpha, indicating onset of spontaneous resolution. Treatment limited PKCzeta cleavage, with expression of nuclear NF-kappaB/p50 and BCL-3 and low nuclear NF-kappaB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCzeta by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-alpha, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-kappaB/p65, increased transforming growth factor-beta2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3.

RATIONALE: NLRs (nucleotide oligomerisation domain [NOD] proteins containing a leucine-rich repeat) are cytosolic pattern recognition receptors. NOD1 senses diaminopimelic acid-containing peptidoglycan present in gram-negative bacteria, whereas NOD2 senses the muramyl dipeptide (MDP) present in most organisms. Bacteria are the most common cause of septic shock, which is characterized clinically by hypotension resistant to vasopressor agents. In animal models, gram-negative septic shock is mimicked by lipopolysaccharide (LPS), which signals through Toll-like receptor 4 (TLR4) and its adaptor MyD88. The role of NLRs in the pathophysiology of septic shock is not known. OBJECTIVES: To compare the effects of selective NOD1 agonists with LPS in vivo. METHODS: Vascular smooth muscle cells or whole aortas from wild-type or genetically modified mice were stimulated in vitro with agonists of NOD1 (FK565) or NOD2 (MDP). Vasoconstriction was measured using wire myography. Nitric oxide (NO) formation was measured using Griess reaction and NO synthase-II protein by Western blotting. In vivo, blood pressure, heart rate, and urine output were measured in sham-, LPS-, or FK565-treated animals. Biomarkers of end-organ injury, coagulation activation, NO, and cytokines were measured in plasma. MAIN RESULTS: FK565, but not MDP, induced NO synthase-II protein/activity in vascular smooth muscle and vascular hyporeactivity to pressor agents. FK565 had no effect on vessels from NOD1(-/-) mice, but was active in vessels from TLR4(-/-), TLR2(-/-), or MyD88(-/-) mice. FK565 induced hypotension, increased heart rate, and caused multiple (renal, liver) injury and dysfunction in vivo. CONCLUSIONS: Activation of NOD1 induces shock and multiple organ injury/dysfunction.

International audience; There is growing evidence that vascular arginase plays a role in pathophysiology of vascular diseases. We recently reported high arginase activity/expression in young adult hypertensive spontaneously hypertensive rats (SHR). The aim of the present study was to characterize the time course of arginase pathway abnormalities in SHR and to explore the contributing role of hemodynamics and inflammation. Experiments were conducted on 5, 10, 19 and 26-week-old SHR and their age-matched control Wistar Kyoto (WKY) rats. Arginase activity as well as expression of arginase I, arginase II, endothelial and inducible NOS were determined in aortic tissue extracts. Levels of L-arginine, NO catabolites and IL-6 (a marker of inflammation) were measured in plasma. Arginase activity/expression was also measured in 10-week-old SHR previously treated with hydralazine (20 mg/kg/day, per os, for 5 weeks). As compared to WKY, SHR exhibited high vascular arginase I and II expression from prehypertensive to established stages of hypertension. However, a mismatch between expression and activity was observed at the prehypertensive stage. Arginase expression was not related either to plasma IL-6 levels or to expression of NOS. Prevention of hypertension by hydralazine significantly blunted arginase upregulation and restored arginase activity. Importantly, arginase activity and blood pressure (BP) correlated in SHR. In conclusion, our results demonstrate that arginase upregulation precedes blood pressure rising and identify elevated blood pressure as a contributing factor of arginase dysregulation in genetic hypertension. They also demonstrated a close relationship between arginase activity and BP, thus making arginase a promising target for antihypertensive therapy.

International audience; Although potentially implicated in several physiological functions, few functional mutations have been identified in the human 5-hydroxytryptamine (HT)(2B) receptor gene. A heterozygous mutation R393X in the 5-HT(2B) receptor was recently identified in a patient diagnosed with pulmonary hypertension after intake of the anorexigenic dexfenfluramine. Although reported to generate a lack of function, this C terminus-truncated 5-HT(2B) receptor should somehow affect transduction pathways relevant to pulmonary hypertension. In our study, we investigated putative modifications in transduction of the R393X-mutated 5-HT(2B) receptor. In stably transfected cells, we confirmed the loss of inositol 1,4,5-trisphosphate stimulation caused by the G(alphaq) uncoupling, despite conserved ligand affinity between the normal and mutated receptors. We also observed a partial loss of nitric-oxide synthase stimulation. However, the truncated R393X receptor presented 1) a strong gain of efficacy in cell proliferation as assessed by mitogen-activated protein kinase activity and thymidine incorporation, 2) a preferential coupling to G(alpha13) as shown by blocking antiserum, and 3) an apparent lack of internalization upon agonist stimulation as observed by confocal microscopy. This work demonstrates that, in the 5-HT(2B) receptor, the C terminus, including the palmitoylation and phosphorylation sites, is absolutely required for proper transduction and internalization. For the first time, we show that the lack of C terminus can generate a switch of coupling to G(alpha13), a reduced NO synthase activation, and an increase in cell proliferation. All these modifications are relevant in pathophysiological vasoconstriction.

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes., Facultad de Ciencias Exactas

International audience; Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-alpha that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the co-immunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. We conclude that activation of AMPK by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex.

International audience; Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

International audience; Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

International audience; Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

International audience; Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

International audience; Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

International audience; Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

International audience; Nitric oxide originating from the endothelial cells of the vessel wall is essential for the vascular system. It is produced by the enzyme endothelial nitric oxide synthase (eNOS). Cellular eNOS activity is affected by changes in eNOS synthesis. To address whether degradation also contributes to eNOS activity, the effect of proteasome inhibitors on eNOS-mediated NO synthesis was studied in the microvascular endothelial cell line bEnd.3 and in cultured primary aortic endothelial cells. Surprisingly, agonist-induced increases in eNOS activity were reduced to 42 and 50% in the presence of the proteasome inhibiting drugs MG132 and clasto-lactacystin-beta-lactone, respectively (P < 0.01). The decrease in activity occurred within 1 hour of drug treatment and was not accompanied by a change in intracellular levels of either eNOS or its inhibitor caveolin-1. Taken together, these data may indicate that eNOS is regulated by an interacting protein, different from caveolin-1, that inhibits its activity and is rapidly degraded by the proteasome in the presence of eNOS agonists.

International audience; Inducible nitric oxide synthase (iNOS) protects heart against ischemia/reperfusion injury. However, it is unknown whether the beneficial effects of iNOS are mediated by the interaction of NO with radical oxygen species (ROS). To address this issue, we examined the effects of liposoluble iron-induced ROS generation in isolated perfused hearts from rats treated with lipopolysaccharide (LPS). LPS administration (10 mg/kg, i.p., 6 h before heart removal) induced iNOS expression and increased NO production as indicated by a 3-fold elevation of nitrite level in coronary effluents relative to control hearts. An enhanced expression of hemeoxygenase 1 protein was also observed in septic hearts compared to control. Iron-induced perfusion and contractile deficits were ameliorated by LPS with more important coronary than myocardial benefits. In iron-loaded hearts, oxidative stress as measured by the 2,3 dihydroxybenzoic acid/salicylic acid concentration ratio in cardiac tissue was 23% lower in septic than in control heart although the difference did not reach significance. In addition, the presence of the NO synthase inhibitor N-nitro-L-arginine in the perfusion medium totally blocked NO production but did not reverse the protective effects of LPS. The results indicate that LPS protects from iron-induced cardiac dysfunction by mechanisms independent on ex vivo NO production and suggest that NO acts as a trigger rather than a direct mediator of the cardioprotective effects of LPS in heart exposed to iron.

International audience; OBJECTIVE: The inducible isoform of nitric oxide synthase (iNOS) is known to be a trigger of the heat stress (HS)-induced cardioprotection. Since iNOS also appears to mediate various forms of myocardial preconditioning, the goal of this study was to investigate its role as a mediator of the HS response. METHODS AND RESULTS: Male Wistar rats were divided in six groups, subjected or not to HS (42 degrees C internal temperature, for 15 min). Twenty-four hours later, they were treated or not with either L-NAME, a non-selective inhibitor of NO synthase isoforms, or 1400W, a selective iNOS inhibitor, 10 min before being subjected to a 30-min left coronary artery occlusion followed by a 120-min reperfusion, in vivo. The infarct size (tetrazolium staining) reducing effect conferred by heat stress (from 46.0+/-1.4% in sham to 26.8+/-3.8% in HS groups) was completely abolished by both L-NAME (53.9+/-3.1%) and 1400W (51.8+/-3.3%). Additional studies using Western blot analysis demonstrated a 3.8-fold increase in myocardial iNOS protein expression 24 h after HS. CONCLUSION: These results suggest an involvement of iNOS as a mediator of the protection conferred by heat stress against myocardial ischaemia.

International audience; In order to investigate the function of haem oxygenase in neuronal cell death or survival, we have determined in PC12 cells whether induction of haem oxygenase mRNA and protein or inhibition of haem oxygenase activity may be able to modulate the cell response to an oxidative stress. Inhibition of glutathione biosynthesis by buthionine sulfoximine (BSO) has indeed been demonstrated, in this cell line, to decrease the intracellular content of glutathione and to trigger a gradual and programmed cell death. Inhibition of haem oxygenase by zinc protoporphyrin IX, a potent inhibitor of this enzyme, or by a recently described peptidic inhibitor, induced a significant decrease in the toxicity of BSO. This protective action was not due to an alteration in the metabolism of glutathione and was still observed when the protecting agent was added several hours after BSO treatment. Induction of haem oxygenase-1 mRNA and protein by either haemin or pyrrolidine dithiocarbamate was associated with no protection or a significant reduction in the toxicity of BSO respectively. Our results indicate that induction of haem oxygenase-1 is not obligatorily associated with an improved resistance towards oxidative stress and suggest that a byproduct of haem degradation may also become detrimental.

International audience; Excess of nitric oxide (NO) has been shown to exert neurotoxic impacts in the brain. Moreover, inhibition of two NO-synthesizing enzymes, neuronal NOS (nNOS) and inducible NOS (iNOS), displays neuroprotective effects in the MPTP model of Parkinson's disease (PD). These data suggest a possible involvement of NOS as factors controlling the resistance of the nigral dopaminergic neurons to environmental insults. Therefore, we investigated whether polymorphisms present in these genes could contribute to the risk of developing PD. We carried out a community-based case-control study among subjects enrolled in the Mutualit?ociale Agricole, the French health insurance organization for workers connected to agriculture. Two-hundred and nine PD patients and 488 controls of European (mostly French) ancestry and matched for age, sex and region of residency were included in this study. Associations were observed with polymorphisms present in exon 22 of iNOS (OR for AA carriers=0.50, 95% CI=0.29-0.86, P=0.01) and in exon 29 of nNOS (OR for carriers of the T allele=1.53, 95% CI=1.08-2.16, P=0.02); no association was observed with a polymorphism in exon 18 of nNOS (OR for carriers of the T allele=1.20, 95% CI=0.85-1.69, P=0.30). Moreover, a significant interaction of the nNOS polymorphisms with current and ever cigarette smoking was found (nNOS 18, P=0.05; nNOS 29, P=0.04). All together, these data favour an involvement of these two genes as new modifier genes in PD.

PARP‐1‐deficient mice display a severe defect in the base excision repair pathway leading to radiosensitivity and genomic instability. They are protected against necrosis induced by massive oxidative stress in various inflammatory processes. Mice lacking p53 are highly predisposed to malignancy resulting from defective cell cycle checkpoints, resistance to DNA damage‐induced apoptosis as well as from upregulation of the iNOS gene resulting in chronic oxidative stress. Here, we report the generation of doubly null mutant mice. We found that tumour‐free survival of parp‐1 −/− p53 −/− mice increased by 50% compared with that of parp‐1 +/+ p53 −/− mice. Tumour formation in nude mice injected with oncogenic parp‐1 −/− p53 −/− fibroblasts was significantly delayed compared with parp‐1 +/+ p53 −/− cells. Upon γ‐irradiation, a partial restoration of S‐phase radiosensitivity was found in parp‐1 −/− p53 −/− primary fibroblasts compared with parp‐1 +/+ p53 −/− cells. In addition, iNOS expression and nitrite release were dramatically reduced in the parp‐1 −/− p53 −/− mice compared with parp‐1 +/+ p53 −/− mice. The abrogation of the oxydated status of p53 −/− cells, due to the absence of parp‐1 , may be the cause of the delay in the onset of tumorigenesis in parp‐1 −/− p53 −/− mice.

Footnote 1:Abbreviations: ACT D, actinomycine D; CHX, cycloheximide; COX, cyclooxygenase; CuDips, Cu(II) (3,5-diisopropylsalicylate)2; DMSO, dimethyl sulfoxide; D-NMMA, Nω-monomethyl-D-arginine; FCS, heat-inactivated fetal calf serum; H2O2, hydrogen peroxide; IL-1β, interleukin-1β; L-Arg, L-arginine; L-NMMA, Nω-monomethyl-L-arginine; NO, Nitric oxide; NO2−, nitrites; NOS, nitric oxide synthase; NSAID, non steroidal anti-inflammatory drug; PGE2, prostaglandin E2; RT-PCR, reverse transcription-polymerase chain reaction; SIN-1, 3-morpholinosydnonimine; SMT, S-methylisothiourea; SOD, superoxide dismutase.; International audience; Cross-talk between inducible nitric oxide synthase (NOS II) and cyclooxygenase-2 (COX-2) was investigated in rat chondrocytes. In monolayers, interleukin-1beta (IL-1beta) induced COX-2 and NOS II expression in a dose- and time-dependent manner, to produce high prostaglandin E(2) (PGE(2)) and nitrite (NO(2)(-)) levels in an apparently coordinated fashion. COX-2 mRNA was induced earlier (30 min. versus 4 hr) and less markedly (4-fold versus 12-fold at 24 hr) than NOS II, and was poorly affected by the translational inhibitor cycloheximide (CHX). IL-1beta did not stabilize COX-2 mRNA in contrast to CHX. Indomethacin and NS-398 lacked any effect on NO(2)(-) levels whereas L-NMMA and SMT reduced PGE(2) levels at concentration inhibiting NO(2)(-) production from 50 to 90%, even when added at a time allowing a complete expression of both enzymes (8 hr). Basal COX activity was unaffected by NO donors. The SOD mimetic, CuDips inhibited COX-2 activity by more than 75% whereas catalase did not. Inhibition of COX-2 by CuDips was not sensitive to catalase, consistent with a superoxide-mediated effect. In tridimensional culture, IL-1beta inhibited radiolabelled sodium sulphate incorporation while stimulating COX-2 and NOS II activities. Cartilage injury was corrected by L-NMMA or CuDips but not by NSAIDs, consistent with a peroxynitrite-mediated effect. These results show that in chondrocytes: (i) COX2 and NOS II genes are induced sequentially and distinctly by IL-1beta; (ii) COX-1 and COX-2 activity are affected differently by NO-derived species; (iii) peroxynitrite accounts likely for stimulation of COX-2 activity and inhibition of proteoglycan synthesis induced by IL-1beta.

International audience; Cross-talk between inducible nitric oxide synthase (NOS II) and cyclooxygenase-2 (COX-2) was investigated in rat chondrocytes. In monolayers, interleukin-1beta (IL-1beta) induced COX-2 and NOS II expression in a dose- and time-dependent manner, to produce high prostaglandin E(2) (PGE(2)) and nitrite (NO(2)(-)) levels in an apparently coordinated fashion. COX-2 mRNA was induced earlier (30 min. versus 4 hr) and less markedly (4-fold versus 12-fold at 24 hr) than NOS II, and was poorly affected by the translational inhibitor cycloheximide (CHX). IL-1beta did not stabilize COX-2 mRNA in contrast to CHX. Indomethacin and NS-398 lacked any effect on NO(2)(-) levels whereas L-NMMA and SMT reduced PGE(2) levels at concentration inhibiting NO(2)(-) production from 50 to 90%, even when added at a time allowing a complete expression of both enzymes (8 hr). Basal COX activity was unaffected by NO donors. The SOD mimetic, CuDips inhibited COX-2 activity by more than 75% whereas catalase did not. Inhibition of COX-2 by CuDips was not sensitive to catalase, consistent with a superoxide-mediated effect. In tridimensional culture, IL-1beta inhibited radiolabelled sodium sulphate incorporation while stimulating COX-2 and NOS II activities. Cartilage injury was corrected by L-NMMA or CuDips but not by NSAIDs, consistent with a peroxynitrite-mediated effect. These results show that in chondrocytes: (i) COX2 and NOS II genes are induced sequentially and distinctly by IL-1beta; (ii) COX-1 and COX-2 activity are affected differently by NO-derived species; (iii) peroxynitrite accounts likely for stimulation of COX-2 activity and inhibition of proteoglycan synthesis induced by IL-1beta.

International audience; Nitric oxide (NO) donors are known to induce both delayed cardioprotection and myocardial heat stress protein (HSP) expression. Moreover, heat stress (HS), which also protects myocardium against ischaemic damages, is associated with a NO release. Therefore, we have investigated the implication of NO in HS-induced resistance to myocardial infarction, in the isolated rat heart model. Rats were divided in six groups (n=10 in each group), subjected or not to heat stress (42 degrees C internal temperature, 15 min) and treated or not with nitro-L-arginine-methylester (L-NAME) a non-selective inhibitor of NO synthase isoforms, or L-N(6)-(1-imino-ethyl)lysine (L-NIL), a selective inhibitor of the inducible NO synthase. Twenty-four hours after heat stress, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Infarct-to-risk ratio was significantly reduced in HS (18.7+/-1.6%) compared to Sham (33.0+/-1.7%) hearts. This effect was abolished in L-NAME-treated (41.7+/-3.1% in HS+L-NAME vs 35.2+/-3.0% in Sham+L-NAME ) and L-NIL-treated (36.1+/-3.4% in HS+L-NIL vs 42.1+/-4.6% in Sham+L-NIL) groups. Immunohistochemical analysis of myocardial HSP 27 and 72 showed an HS-induced increase of these proteins, which was not modified by L-NAME pretreatment. We conclude that NO synthases, and in particular the inducible isoform, appear to play a role in the heat stress-induced cardioprotection, independently of HSP 27 and 72 levels. Further investigations are required to elucidate the precise role of HSPs in this adaptive response.

Poly (ADP-ribose) polymerase-1 is a nuclear DNA-binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP-1-deficient cells are defective in NF-kappaB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-alpha. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF-kappaB in macrophages from PARP-1(+/+) but not from PARP-1(-/-) mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-kappaB-dependent accumulation of TNF-alpha in the serum and a down-regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-kappaB, with consequences for the transcriptional activation of NF-kappaB and a systemic inflammatory process.

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-gamma and MyD88 molecules with a partial contribution of TNF-alpha and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice., info:eu-repo/semantics/published

International audience; The enzyme endothelial nitric oxide synthase (eNOS) is essential for vascular integrity. Many studies have demonstrated a link between the localization and activity of eNOS. Here, we studied the influence of cell-cell contact on this link in the microvascular endothelial bEnd.3 cell line. By immunofluorescence microscopy, eNOS localization at the plasma membrane was found to be dependent on cell-cell contact. In particular, eNOS was highly enriched at the intercellular contact sites. Further analysis showed that the pattern of eNOS localization at the plasma membrane resembled that of PECAM-1 (platelet endothelial cell adhesion molecule 1), but not that of the adherens junction proteins VE (vascular endothelial)-cadherin and plakoglobin. eNOS that was localized at the contact sites was, in part, Triton X-100-insoluble, in contrast with eNOS at the Golgi complex, which may indicate an association of eNOS with the actin cytoskeleton. Interestingly, eNOS activity was up-regulated in confluent monolayers compared with subconfluent cells, while there was no difference in eNOS expression. This correlation between cell confluence and eNOS activity was also found when primary bovine aortic endothelial cells were studied. These data imply that cell-cell contact induces the localization of eNOS at intercellular junctions, which is required for agonist-induced eNOS activation.

International audience; Our aim was to investigate the role of oxidative stress in elastase-induced pulmonary emphysema. C57BL/6 mice were subjected to pancreatic porcine elastase (PPE) instillation (0.05 or 0.5 U per mouse, i.t.) to induce pulmonary emphysema. Lungs were collected on days 7, 14, and 21 after PPE instillation. The control group was sham injected. Also, mice treated with 1% aminoguanidine (AMG) and inducible NO synthase (iNOS) knockout mice received 0.5 U PPE (i.t.), and lungs were analyzed 21 days after. We performed bronchoalveolar lavage, biochemical analyses of oxidative stress, and lung stereology and morphometry assays. Emphysema was observed histologically at 21 days after 0.5 U PPE treatment; tissues from these mice exhibited increased alveolar linear intercept and air-space volume density in comparison with the control group. TNF-α was elevated at 7 and 14 days after 0.5 U PPE treatment, concomitant with a reduction in the IL-10 levels at the same time points. Myeloperoxidase was elevated in all groups treated with 0.5 U PPE. Oxidative stress was observed during early stages of emphysema, with increased nitrite levels and malondialdehyde and superoxide dismutase activity at 7 days after 0.5 U PPE treatment. Glutathione peroxidase activity was increased in all groups treated with 0.5 U PPE. The emphysema was attenuated when iNOS was inhibited using 1% AMG and in iNOS knockout mice. Furthermore, proteolytic stimulation by PPE enhanced the expression of nitrotyrosine and iNOS, whereas the PPE+AMG group showed low expression of iNOS and nitrotyrosine. PPE stimulus also induced endothelial (e) NOS expression, whereas AMG reduced eNOS. Our results suggest that the oxidative and nitrosative stress pathways are triggered by nitric oxide production via iNOS expression in pulmonary emphysema.

International audience; H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.

Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in the avascular retina, mediated at least in part by a downregulation of VEGF receptor 2 (VEGFR2) in cells adjacent to iNOS-expressing cells. At the same time, pathological intravitreal neovascularization is considerably stronger in iNOS-expressing animals. These findings demonstrate that iNOS plays a crucial role in retinal neovascular disease and show that it offers an ideal target for the control of vitreal neovascularization through improvement of the vascularization of the hypoxic retina.