Basal NO Production Research Articles

Repurposing of monocyclic β-lactams as anti-inflammatory agents – The case of new ferrocene-azetidin-2-one hybrids

There is growing interest in developing monotherapy drugs that treat inflammation caused by microbial infections, focusing on dual antimicrobial and anti-inflammatory agents with minimal side effects and high safety margins. This study synthesized and characterized a library of novel cis-4-ferrocenylazetidin-2-ones, evaluating their antimicrobial and anti-inflammatory activities. These organometallic monocyclic β-lactams showed moderate in vitro antimicrobial activity against various standard microbial strains, including yeasts and Gram-negative and Gram-positive bacteria. Some compounds overcame the resistance of clinical Staphylococcus aureus isolates. Traditionally, monocyclic β-lactams target Gram-negative bacilli, but adding a ferrocene moiety and substituting the COOH group near the N-1 position with a non-ionizable ester group (COOR) extended their activity spectrum. The anti-inflammatory properties were assessed in macrophage-based models, revealing non-cytotoxicity below 10 μM. Two compounds were shown to be strong and selective arginase inhibitors, while five others effectively suppressed excessive NO formation without affecting basal NO production. The presence of a phenoxy group at C-3 of the β-lactam ring appeared to be crucial for selective NO inhibition. These hybrids did not scavenge NO but inhibited NO synthesis by suppressing iNOS expression. Overall, two novel hybrids were identified as promising hit candidates for treating infection-induced inflammatory reactions.

Reduced microvascular flow-mediated dilation in Syrian hamsters lacking d-sarcoglycan is caused by increased oxidative stress.

d-sarcoglycan mutation reduces mechanotransduction and induces dilated cardiomyopathy with aging. We hypothesized that in young hamsters with d-sarcoglycan mutation, which do not show cardiomyopathy, flow mechanotransduction might be affected in resistance arteries as the control of local blood flow. Flow-mediated-dilation (FMD) was measured in isolated mesenteric resistance arteries, using 3-months old hamsters carrying a mutation in the d-sarcoglycan gene (CH-147) and their control littermates. The FMD was significantly reduced in the CHF-147 group. Nevertheless, passive arterial diameter, vascular structure and endothelium-independent dilation to sodium nitroprusside were not modified. Contraction induced by KCl was not modified, whereas contraction due to phenylephrine was increased. The basal NO production and total eNOS expression levels were not altered. Nevertheless, eNOS phosphorylation, FAKs and RhoA expression were reduced in CH-147. In contrast, p47phox, COX2, iNOS and reactive oxygen species levels were higher in the endothelium of CHF-147 hamsters. Reducing ROS levels using the superoxide dismutase analog Tempol significantly restored the flow-mediated dilation (FMD) levels in CHF-147 hamsters. However, treatment with the COX-2 inhibitor NS-398 showed a non-significant improvement in FMD.

The effect of hyperglycemia on the activation of peritoneal macrophages of albino rats.

Hyperglycemia is one of the main damaging factors of diabetes mellitus (DM). The severity of this disease is most clearly manifested under conditions of the inflammatory process. In this work, we have studied the activation features of rat peritoneal macrophages (MPs) under conditions of high glucose concentration in vitro. Comparison of the independent and combined effects of streptozotocin-induced DM and hyperglycemia on proliferation and accumulation of nitrites in the MPs culture medium revealed similarity of their effects. Elevated glucose levels and, to a lesser extent, DM decreased basal proliferation and NO production by MPs in vitro. The use of the protein kinase C (PKC) activator, phorbol ester (PMA), abolished the proinflammatory effect of thrombin on PMs. This suggests the involvement of PKC in the effects of the protease. At the same time, the effect of thrombin on the level of nitrites in the culture medium demonstrates a pronounced dose-dependence, which was not recognized during evaluation of proliferation. Proinflammatory activation of MPs is potentiated by hyperglycemia, one of the main pathological factors of diabetes. Despite the fact that high concentrations of glucose have a significant effect on proliferation and NO production, no statistically significant differences were found between the responses of MPs obtained from healthy animals and from animals with streptozotocin-induced DM. This ratio was observed for all parameters studied in the work, during analysis of cell proliferation and measurement of nitrites in the culture medium. Thus, the results obtained indicate the leading role of elevated glucose levels in the regulation of MPs activation, which is comparable to the effect of DM and even "masks" it.

Endothelial Contribution to Warfarin-Induced Arterial Media Calcification in Mice.

Arterial media calcification (AMC) is predominantly regulated by vascular smooth muscle cells (VSMCs), which transdifferentiate into pro-calcifying cells. In contrast, there is little evidence for endothelial cells playing a role in the disease. The current study investigates cellular functioning and molecular pathways underlying AMC, respectively by, an ex vivo isometric organ bath set-up to explore the interaction between VSMCs and ECs and quantitative proteomics followed by functional pathway interpretation. AMC development, which was induced in mice by dietary warfarin administration, was proved by positive Von Kossa staining and a significantly increased calcium content in the aorta compared to that of control mice. The ex vivo organ bath set-up showed calcified aortic segments to be significantly more sensitive to phenylephrine induced contraction, compared to control segments. This, together with the fact that calcified segments as compared to control segments, showed a significantly smaller contraction in the absence of extracellular calcium, argues for a reduced basal NO production in the calcified segments. Moreover, proteomic data revealed a reduced eNOS activation to be part of the vascular calcification process. In summary, this study identifies a poor endothelial function, next to classic pro-calcifying stimuli, as a possible initiator of arterial calcification.

Individual red blood cell nitric oxide production in sickle cell anemia: Nitric oxide production is increased and sickle shaped cells have unique morphologic change compared to discoid cells

Sickle cell anemia (SCA) is characterized by decreased red blood cell (RBC) deformability due to polymerization of deoxygenated hemoglobin, leading to abnormal mechanical properties of RBC, increased cellular adhesion, and microcirculatory obstruction. Prior work has demonstrated that NO• influences RBC hydration and deformability and is produced at a basal rate that increases under shear stress in normal RBC. Nevertheless, the origin and physiological relevance of nitric oxide (NO•) production and scavenging in RBC remains unclear. We aimed to assess the basal and shear-mediated production of NO• in RBC from SCA patients and control (CTRL) subjects. RBCs loaded with a fluorescent NO• detector, DAF-FM (4-Amino-5-methylamino- 2′,7′-difluorofluorescein diacetate), were imaged in microflow channels over 30-min without shear stress, followed by a 30-min period under 0.5Pa shear stress. We utilized non-specific nitric oxide synthase (NOS) blockade and carbon monoxide (CO) saturation of hemoglobin to assess the contribution of NOS and hemoglobin, respectively, to NO• production. Quantification of DAF-FM fluorescence intensity in individual RBC showed an increase in NO• in SCA RBC at the start of the basal period; however, both SCA and CTRL RBC increased NO• by a similar quantity under shear. A subpopulation of sickle-shaped RBC exhibited lower basal NO• production compared to discoid RBC from SCA group, and under shear became more circular in the direction of shear when compared to discoid RBC from SCA and CTRL, which elongated. Both CO and NOS inhibition caused a decrease in basal NO• production. Shear-mediated NO• production was decreased by CO in all RBC, but was decreased by NOS blockade only in SCA. In conclusion, total NO• production is increased and shear-mediated NO• production is preserved in SCA RBC in a NOS-dependent manner. Sickle shaped RBC with inclusions have higher NO• production and they become more circular rather than elongated with shear.

Abstract 069: Local and Circulating Sphingosine-1-Phosphate in Blood Pressure Regulation

Sphingosine-1-phosphate (S1P), a bioactive lipid, regulates different biological processes in health and disease. Once synthetized, endothelial S1P is transported outside the cells via Spinster-2 (Spns-2), and can signal mainly through the sphingosine-1-phosphate receptor-1 (S1P1) in autocrine/paracrine manner. Alternatively, S1P can bind circulating carriers, including ApoM of HDL or albumin. Recently, we reported a key role of endothelial S1P-S1P1 autocrine signaling in vascular function and blood pressure (BP) homeostasis. To dissect the contribution of endothelium-derived S1P versus circulating HDL-bound S1P on BP regulation, we used ApoM knockout mice (ApoM-/-) and developed mice lacking the endothelial Spinster-2 (ECKO-Spns2). At baseline, systolic BP (SBP) was markedly increased in both ECKO-Spns2 (120.8± 2.1 vs. 106.6±1.3 mmHg, n=8) and ApoM-/- (122.6±1.0 vs. 109.0±1.4 mmHg, n=8) vs control mice. Vasodilation of mesenteric arteries (MA) to acetylcholine was preserved in both mouse models, whereas flow-induced vasodilation was significantly reduced in ECKO-Spns2 (Emax 36.0±1.5 vs. 53.6±3.6 % vasodilation, n=5) but not in ApoM-/- MA, suggesting that autocrine S1P signaling requires the S1P transporter Spns-2. However, both ECKO-Spns2 and ApoM-/- showed a reduced NO production compared to control MA. Moreover, chronic infusion of AngII resulted in higher BP in ECKO-Spns2 (156.0± 1.2 vs. 142.0±1.6 mmHg, n=8) and ApoM-/- (158.5±1.1 vs. 144.1±1.3 mmHg, n=8) vs control mice. Interestingly, chronic AngII strongly reduced vasodilation in response to flow in both ECKO-Spns2 (Emax 19.3±2.5 vs. 35.4±7.1 % vasodilation, n=5) and ApoM-/- (Emax 22.9±1.1 vs. 34.8±6.8 % vasodilation, n=5) compared to control MA, suggesting a protective role of local and circulating S1P. Additionally, basal NO production was further impaired by the AngII infusion (ECKO-Spns2 -32.7% and ApoM-/- -42.8%, vs control MA), confirming the pivotal role of S1P-S1P1-NO pathway in vascular and BP homeostasis.

Light-induced formation of NO in endothelial cells by photoactivatable NADPH analogues targeting nitric-oxide synthase

BackgroundNitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2′- or/and 3′-position(s) of the ribose in the adenosine moiety, instead of a 2′-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells. MethodsThe cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively. ResultsWe found that, two compounds, those bearing a single carboxymethyl group on the 3′-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects. ConclusionsWe show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells. General significanceEfficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes.

Chemerin/CMKLR1 axis influences pulmonary arterial pressure and the NO signaling pathway

Pulmonary arterial hypertension (PAH) is a clinical condition associated with sustained elevations in pulmonary arterial pressure, leading to right ventricular failure. The adipokines leptin and adiponectin have shown respectively deleterious and protective effects in animal models of PAH. Chemerin is a novel adipokine implicated in blood pressure regulation and recently associated with atherosclerosis, essential hypertension and obesity-induced hypertension. In this context, we aimed to determine whether the chemerin/CMKLR1 axis could influence pulmonary arterial pressure and the severity of pulmonary hypertension. Age-matched wild-type (WT) and CMKLR1 knockout (KO) mice were used. We localized chemerin and the chemokine-like receptor-1 (CMKLR1) in lung sections, pulmonary artery and thoracic aorta. WT and KO mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks ( N = 6 per group). Right ventricular systolic pressure (RVSP) was measured with a micro-tip catheter. Segments of pulmonary artery and thoracic aorta were incubated, immediately after sacrifice, with recombinant mouse chemerin (10–8 mol/L, 1 hour) or vehicle, then stimulated or not with acetylcholine (ACH) (10–4 mol/L, 5 min) to measure NO production in supernatants by determination of nitrites. RVSP was higher in KO than in WT mice in normoxia (26.6 ± 0.4 vs. 22.2 ± 1 mmHg, P = 0.001) and in hypoxia (37.8 ± 0.5 vs. 31.4 ± 1.2 mmHg, P = 0.001). Hypoxia reduced NO production in WT and KO mice. In WT, ACH-induced NO production was abolished by chemerin pre-incubation. In normoxia and hypoxia, basal NO production was lower in KO than WT mice and ACH-stimulated NO production was totally inhibited in KO mice. These observations provide support for a role of the chemerin/CMKLR1 axis as regulator of pulmonary vascular homeostasis and as a disease-modifier in the context of pulmonary hypertension by the negative modulation of pulmonary vascular NO production.

Zinc Modulates Endothelin-1 and Nitric Oxide Signalling in Vascular Endothelial and Smooth Muscle Cells

Zn homeostasis is an important regulator of vascular function, from the inflammatory response to vasodilation. Smoking significantly decreases endothelial Zn levels and causes loss of ZIP2 (Zn cystosolic influx transporter) expression in mice. Endothelin-1 (ET1), an endothelium-derived vasoconstricting factor, is systemically elevated in smokers, who also have lowered Zn, impaired NO production and can present increased risk of coronary vasospasm. Furthermore, circulating ET1 levels are elevated in patients with coronary slow flow; a microvascular disorder with higher prevalence in smokers. Our preliminary data have shown Zn chelation caused vasoconstriction of human subcutaneous microvessels and ET1-dependent vasoconstriction was significantly ablated by physiological Zn levels in a concentration dependent manner. On this basis, we extended this study to see which Zn transporters (ZnT1-10 and ZIP1-14) respond to changing Zn homeostasis in endothelial (EC) and smooth muscle cells (SMC), and the effect of Zn on ET1 and NO signalling. In both EC and SMC, ZIP2 and ZIP12 gene expression were upregulated under Zn depleted conditions (20 uM TPEN; n = 4). Preliminary data show that both basal NO production and intracellular ET1 levels were increased in EC with increasing intracellular Zn concentrations. However, intracellular ET1 protein was markedly reduced under Zn depleted conditions in EC, while it was increased in SMC (fluorescence confocal microscopy). Hypozincaemia may therefore trigger ET1 release, which may reflect a coupled role for Zn and NO in opposing ET1 release. This will be confirmed by measuring ET1 in the extracellular supernatant.

Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation.

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

Influence of Apoptotic Bodies and Apoptotic Microvesicles on NO Production in Macrophages.

We studied the effect of extracellular vesicular particles generated during apoptosis by macrophages of M0, M1 and M2 phenotypes on spontaneous and LPS-stimulated production of NO. The fractions of apoptotic bodies and apoptotic microvesicles were obtained in the primary cultures of peritoneal macrophages undergoing apoptosis. The effect of these microparticles on LPS-induced proinflammatory response of recipient macrophages critically depends on the initial phenotype of "donor" macrophages. Microvesicles and especially apoptotic bodies from M1 macrophages stimulate basal NO production. LPS stimulation of these macrophages preincubated with apoptotic bodies was not followed by further growth of NO production; in macrophages preincubated with microvesicles, LPS even suppressed NO production. Apoptotic microparticles obtained from M2 macrophages produced little effect on the basal production of NO. LPS stimulation of macrophages-recipients preincubated with microparticles from M2 macrophages did not enhance NO production. Incubation of macrophages with apoptotic microparticles induces the formation of endotoxic tolerance.

VASCULAR NO AND SUPROXIDE PRODUCTION IS INFLUENCED BY HIGH DIETARY SALT INTAKE IN TFF3−/− MICE

Objective: Arachidonic acid metabolism and products are shown to affect vascular reactivity mechanism. TFF3−/− mice have protective phenotype of favorable ratio of ω6/ω-3 fatty acids and modified metabolism of arachidonic acid. However, the role of TFF3 peptide in hypertension and vascular function is still underinvestigated. The aim of this study was to asses the effect of high salt diet on flow-induced endothelial NO and superoxide production in carotid arteries of wild type mice of mixed background C57Bl6/Sv/129 (WT) and TFF3−/− knock out mice with the same background. Design and method: Eleven weeks old male TFF3−/− or WT mice were divided in 2 groups (N = 4 per group): low salt (LS) and high salt (HS; 4% NaCl for 7 days). Following dietary protocol, mice were anaesthetized with ketamine (75 mg/kg) and midazolam (2.5 mg/kg). Carotid arteries were isolated and cannulated on pressure myograph with or without flow (at delta 80 mmHg), in the absence/presence of the NOS inhibitor L-NAME. NO production was determined by DAF-2DA to DAF-2T conversion fluorescence assay. To assess the production of superoxide radicals, the carotid arteries were loaded with dihydroethidine (DHE, 20 μM). All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by institutional Ethical Committee. p < 0.05 was significant. Results: Basal NO production in no-flow condition was similar among groups. L-NAME blocked the production of NO in all groups. Flow-induced NO production was decreased in HS-WT group compared to no-flow conditions in HS-WT group, and compared to LS-WT group and LS-TFF3−/− group (p < 0.05). There were no changes in endothelial NO production in TFF3−/− mice. Superoxide production was increased in LS-TFF3−/− no-flow group compared to LS-WT in no-flow condition Flow-induced superoxide production was increased in both HS-WT and HS-TFF3−/− groups compared to corresponding LS groups. Conclusions: HS intake decreases flow-induced vascular NO production in WT mice probably due to increased oxidative stress. However, HS intake does not affect NO production in TFF3−/− mice eventhough superoxide levels are increased in basal levels and in flow conditions, suggesting protective role of this phenotype on NO bioavailability.

Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress

The endothelium is exposed to various flow patterns such as vasoprotective unidirectional laminar shear stress (LSS) and atherogenic oscillatory shear stress (OSS). A software-controlled, valve-operated OsciFlow device with parallel chambers was used to apply LSS and OSS to endothelial cells. Although LSS inhibited superoxide over time, OSS time-dependently increased superoxide production from endothelial cells. Immunocytochemical staining revealed that, at resting state, p47phox colocalizes with NOX2, whereas NOXO1 colocalizes with NOX1. RNAi of p47phox had no effects on superoxide or NO production in response to OSS but significantly reduced NO production in LSS, implicating a p47phox-bound NADPH oxidase (NOX) in mediating basal NO production. Indeed, RNAi of p47phox inhibited endothelial nitric oxide synthase (eNOS) serine 1179 phosphorylation, whereas PEG-catalase scavenging of intracellular hydrogen peroxide or RNAi of NOX2 produced similar results, indicating a role of NOX2/p47phox-derived hydrogen peroxide in mediating the basal activity of NO production from eNOS. In contrast, RNAi of NOXO1 resulted in no significant changes in NO and superoxide levels in response to LSS but significantly reduced superoxide while increasing NO in response to OSS. Furthermore, we identified, for the first time, that OSS uncouples eNOS, which was corrected by RNAi of NOXO1. In summary, LSS activates the NOX2-p47phox complex to activate eNOS phosphorylation and NO production. OSS instead activates the NOX1-NOXO1 complex to uncouple eNOS. These results demonstrate differential roles of NOXs in modulating the redox state in response to different shear stresses, which may promote the development of novel therapeutic agents to mimic the protective effects of LSS while inhibiting the injurious effects of OSS.

Mechanisms of vascular dysfunction in acute phase of Trypanosoma cruzi infection in mice

Vascular disorders have a direct link to mortality in the acute phase of Trypanosoma cruzi infection. However, the underlying mechanisms of vascular dysfunction in this phase are largely unknown. We hypothesize that T. cruzi invades endothelial cells causing dysfunction in contractility and relaxation of the mouse aorta. Immunodetection of T. cruzi antigen TcRBP28 was observed in endothelial cells. There was a decreased endothelial nitric oxide synthase (eNOS)-derived NO-dependent vascular relaxation, and increased vascular contractility accompanied by augmented superoxide anions production. Endothelial removal, inhibition of cyclooxygenase 2 (COX-2), blockade of thromboxane A2 (TXA2) TP receptors, and scavenger of superoxide normalized the contractile response. COX-2, thromboxane synthase, inducible nitric oxide synthase (iNOS), p65 NFκB subunit and p22phox of NAD(P)H oxidase (NOX) subunit expressions were increased in vessels of chagasic animals. Serum TNF-α was augmented. Basal NO production, and nitrotyrosine residue expression were increased. It is concluded that T. cruzi invades mice aorta endothelial cells and increases TXA2/TP receptor/NOX-derived superoxide formation. Alongside, T. cruzi promotes systemic TNF-α increase, which stimulates iNOS expression in vessels and nitrosative stress. In light of the heart failure that develops in the chronic phase of the disease, to understand the mechanism involved in the increased contractility of the aorta is crucial.

Effect of treatment with Victoza® on cafeteria induced-obese rats.

Metabolic syndrome is a condition that individuals develop cardiovascular health, diabetes and others complicationsThe dramatic rise in the prevalence of obesity and type 2 diabetes mellitus (T2DM) is associated with increased mortality, morbidity as well as public health care expenses worldwide. The GLP1 is a class of incretins secreted by intestine that improve insulin secretion. The objective was evaluating the effect of treatment with Victoza® on cafeteria induced-obese rats. The rats were divided in two groups: Control (animals that received chow diet) and DIET (animals that to 30th received cafeteria diet until 90th). Ten days before de experimental process the animals was divided in two groups again: CON GLP1 and DIET GLP1 (animals DIET that received subcutaneous injections of Victoza® 0,0077 mg/day during 10 days).The weight of fats are showed in table 1. Fats Normal Con Normal Diet Con GLP1 Diet GLP1 Retroperitoneal fat pad 8,01±0,93* 11,93±1,34 6,14±0,52 12,9±1,36 Periepidydimal fat pad 5,31±0,66* 10,92±1,16 4,66±0,50 10,46±1,36 mesenteric fat pad 4,06±0,46*† 9,64±1,42 2,64±0,33 6,21±1,0 The blood data showed that the cafeteria diet caused increased blood glucose levels, showed in figure 1. The cafeteria diet also promoted increased triglycerides and treatment with Victoza® decreased in the Con GLP1 and Diet GLP1 groups being more significant in the diet group. Preliminary results showed treatment with Victoza® caused an increase in basal NO production of peritoneal macrophage for both control and diet groups, in opposites reduce the basal ROS production. In conclusion ours results showed that treatment of Victoza® causes reduction in glycemia and triglycerides and alters macrophages functions.These findings are of great importance since obesity is the major risk factor for several chronic diseases.

Effects of anti-inflammatory drugs in primary kidney cell culture of a freshwater fish

The non-steroidal anti-inflammatory drugs are emerging contaminants in aquatic ecosystems. This study aimed to evaluate toxic effects of some representative drugs of this pharmaceutical group on primary culture of monocytic lineage of Hoplias malabaricus anterior kidney. The effects of diclofenac, acetaminophen and ibuprofen in cell viability, lipopolysaccharide (LPS)-induced NO production and genotoxicity were evaluated. Cytometry analysis CD11b+ cells showed 71.5% of stem cells, 19.5% of macrophages and 9% of monocytes. Cell viability was lower in the ficoll compared to percoll separation. LPS-induced NO production by these cells was blocked after treatment with dexamethasone and NG-Methyl-L-Arginine (L-NMMA). Exposure of the cells to diclofenac (0.2–200 ng/mL), acetaminophen (0.025–250 ng/mL) ibuprofen (10–1000 ng/mL) reduced basal NO production and inhibited LPS-induced NO production at all concentrations after 24 h of exposure. Genotoxicity occurred at the highest concentration of diclofenac and at the intermediary concentration of acetaminophen. Genotoxicity was also observed by ibuprofen. In summary, the pharmaceuticals influenced NO production and caused DNA damage in monocytic cells suggesting that these drugs can induce immunosuppression and genotoxicity in fish.

Age‐related cerebrovascular endothelial dysfunction is associated with cognitive impairment in C57BL/6 mice

Mild cognitive impairment (MCI) is an age‐related disorder that contributes to a reduced quality of life in older adults. However, the underlying mechanisms contributing to MCI with aging are incompletely understood. We tested the hypothesis that old C57 male mice demonstrate cerebrovascular endothelial dysfunction associated with MCI. Endothelium‐dependent dilation (EDD) (ex vivo dilation of mid cerebral and posterior communication arteries to acetylcholine [ACh]) was impaired in old (O: 22–26 mo, n= 17) vs. young (Y: 3–6 mo, n=11) mice (51.2 ± 5.9 % vs. 72.4 ± 5.4 %, p&lt;0.05), and this was associated with reduced L‐NAME‐sensitive dilation (O: 16.1 ± 9.1 % vs. Y: 49.5 ± 9.5 %, p&lt;0.05). Basal NO inhibition by L‐NAME had no effect on phenylephrine‐induced vasoconstriction in O (Con. 23.1 ± 2.8 % vs. L‐NAME 31.1 ± 4.1 %), but increased constriction in Y (Con 19.9 ± 3.1 % vs. L‐NAME 38.0 ± 3.9 %, p&lt;0.05). O (n = 7) had impaired spatial learning during the Morris water maze test (p&lt;0.05) and this was unrelated to differences in vision. In the probe trial, O had lower platform crossing numbers than Y (n =8) (O: 2.7 ± 0.5 vs. Y: 5.1 ± 0.7, p&lt;0.05), suggesting O had impaired spatial memory compared to Y. These results indicate that cerebrovascular endothelial dysfunction, as reflected by reduced basal NO production and NO‐dependent EDD, develops with aging and is associated with MCI in C57 mice.Work supported by NIH AG013038 and AG000279.

Neuronal nitric oxide synthase regulates endothelial inflammation

NO, produced by the endothelium, is a modulator of vascular inflammation. Traditionally, eNOS was believed to be the primary source of NO in the endothelium. However, recent data suggest an important role for nNOS in the endothelium, although little is known about factors regulating this novel eNOS. We examined the localization, regulation, and significance of endothelial nNOS in this study. Primary HUVECs were used as a model system. Inflammatory changes were induced by stimulation with TNF. We report that unlike eNOS, nNOS is predominantly localized to the nucleus of resting endothelial cells. This nNOS also contributed to basal NO production in the resting endothelium. Ablation of endothelial nNOS by pharmacological inhibition (using L-NPA) or siRNA further enhanced cytokine-mediated inflammatory responses, such as up-regulation of VCAM-1 and proinflammatory cytokines, as well as increased leukocyte recruitment. Based on these findings, we suggest a potential anti-inflammatory role of endothelial nNOS that can attenuate unopposed, proinflammatory cytokine actions. Our data indicate a novel location and an immunoregulatory role for nNOS in the endothelium.

Endothelial Nitric Oxide Synthase Gene Polymorphism and Periodontal Disease

The endothelial isoform of nitric oxide synthase (eNOS) is responsible for the physiological production of NO in endothelial cells and platelets. There is evidence that the G894T polymorphism of the eNOS gene is associated with this enzyme's basal activity and NO production, which could contribute to the pathogenesis of periodontal diseases (PD). Therefore, this study was designed to investigate the role of G894T polymorphism in the eNOS gene as a predisposing factor to periodontal disease. In this study we investigated the association of this polymorphism with PD in an admixture population (N = 119) separated into three groups: Healthy control, Moderate and Severe PD, without statistical differences among them for risk factors for PD, such as age, gender and smoking status. We observed that the GG genotype was associated with the progression of PD as indicated by an increase in frequency of approximately 18% in the Moderate and 26% in the Severe groups compared to the Healthy control group (p = 0.0302). This finding indicates that patients carrying the GG genotype have a greater chance of developing PD compared with those carrying the T allele, and it reinforces the notion that genetic factors contribute to the development and aggravation of PD.

246 Respective roles of transactivating function-1 and -2 of estrogen receptor alpha in the vasculoprotective actions of estradiol

Full length 66 kDa estrogen receptor alpha (ER) stimulates target gene transcription through two activation functions (AF), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46 kDa ER isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. To evaluate the involvement of ER AF-1 and AF-2 in the vasculoprotective actions of estradiol (E2), we generated a targeted deletion of the ER A/B domain in the mouse named ERAF-10 mice, and a targeted deletion of amino acids 543–549 and thus deficient in AF-2 (named ERAF-20 mice). Both basal endothelial NO production was increased by E2 administration in a similar extent than in control mice. E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18 week-old ERAF-1+/+ LDL-r-/- (Low Density Lipoprotein receptor) and ERAF-10 LDL-r-/- mice fed with a hypercholesterolemic diet. We conclude that ER AF-1 is not required for the vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, Selective Estrogen Receptor Modulators stimulating ER independently of the A/B domain and thereby with minimal activation of ER AF-1 could retain beneficial vascular actions, while minimizing the sexual effects. The results concerning ERAF-20 mice are in process and will be available at the end of 2010, and the precise role of AF2 in these actions will be presented.