Nitric Oxide-independent Mechanism Research Articles

Epigenetic regulation by hypoxia, N-acetylcysteine and hydrogen sulphide of the fetal vasculature in growth restricted offspring: A study in humans and chicken embryos.

Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H2S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H2S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H2S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H2S production and enhanced third-order femoral artery dilator responses to the H2S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H2S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H2S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H2S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H2S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy.

Proteomics Studies Suggest That Nitric Oxide Donor Furoxans Inhibit In Vitro Vascular Smooth Muscle Cell Proliferation by Nitric Oxide-Independent Mechanisms.

Physiologically, smooth muscle cells (SMC) and nitric oxide (NO) produced by endothelial cells strictly cooperate to maintain vasal homeostasis. In atherosclerosis, where this equilibrium is altered, molecules providing exogenous NO and able to inhibit SMC proliferation may represent valuable antiatherosclerotic agents. Searching for dual antiproliferative and NO-donor molecules, we found that furoxans significantly decreased SMC proliferation in vitro, albeit with different potencies. We therefore assessed whether this property is dependent on their thiol-induced ring opening. Indeed, while furazans (analogues unable to release NO) are not effective, furoxans' inhibitory potency parallels with the electron-attractor capacity of the group in 3 of the ring, making this effect tunable. To demonstrate whether their specific block on G1-S phase could be NO-dependent, we supplemented SMCs with furoxans and inhibitors of GMP- and/or of the polyamine pathway, which regulate NO-induced SMC proliferation, but they failed in preventing the antiproliferative effect. To find the real mechanism of this property, our proteomics studies revealed that eleven cellular proteins (with SUMO1 being central) and networks involved in cell homeostasis/proliferation are modulated by furoxans, probably by interaction with adducts generated after degradation. Altogether, thanks to their dual effect and pharmacological flexibility, furoxans may be evaluated in the future as antiatherosclerotic molecules.

Raspberry Supplementation Attenuates Blood Pressure and Improves Mesenteric Artery Relaxation in a Nitric Oxide-Independent Mechanism in Angiotensin II-Treated Rats

ObjectivesBerries are rich in polyphenols with strong antioxidant capacity. They have been suggested to exert antihypertensive properties, which may be due to an increase in the levels of endothelial nitric oxide (NO), a potent vasodilator. Thus, the objective of this study was to evaluate the effects of raspberry supplementation on vascular function in angiotensin (Ang) II-treated rats. MethodsEight-week-old Sprague-Dawley rats (n = 8/group) were fed an AIN-93M diet (control, Ang II, and Losartan (LOS; 20 mg/kg/BW) groups) or AIN-93M diet supplemented with 10% w/w freeze-dried raspberry (RB + Ang II) for 7 weeks. At week 5, rats were implanted with subcutaneous osmotic minipumps that delivered 0.9% saline (CON) or Ang II (270 ng/kg BW/min) for an additional 3 weeks. Blood pressure (BP) was measured using tail-cuff plethysmography (n = 7–8/group). First order mesenteric arteries were isolated for vascular relaxation measurements following constriction by U46619 (1 μM) in a wire myograph (n = 3–8/group). Data were found to be normal and analyzed using ANOVA or mixed-model where appropriate. ResultsAng II group showed a significant increase in systolic BP (SBP) (+26.8%, p = 0.002) 2 weeks after implantation as compared to baseline, while CON, RB + Ang II, and LOS groups showed no differences. Final SBP was significantly lower in the CON (–26.4%, p = 0.01), LOS (–28.3%, p = 0.0005), and RB + Ang II (–20.8%, p = 0.03) groups compared to Ang II group. Relaxation was significantly inhibited in the Ang II group in the presence of acetylcholine after incubation with L-NAME (100 μM), a NO synthase inhibitor, when compared to LOS (p = 0.0003) and RB + Ang II (p = 0.0004) groups. Similarly, relaxation in the Ang II group was significantly inhibited after incubation with ODQ (10 μM), a sGC inhibitor, when compared to CON (p = 0.0002), LOS (p = 0.0004), and RB + Ang II (p = 0.0001) group. No significant changes in vessel relaxation were observed after incubation with LY294002 (20 μM), a PI3K inhibitor. ConclusionsWe demonstrate that RB supplementation attenuated Ang II-induced hypertension and abolished the inhibitory effects of L-NAME and ODQ. This suggests that RB may exert its vasodilatory effects in a NO-independent manner. Funding SourcesThe Agriculture and Food Research Initiative (grant no. 2019–67,017-29,257/project accession no. 1,018,642) from the USDA National Institute of Food and Agriculture.

The GPR18 Agonist PSB-KD-107 Exerts Endothelium-Dependent Vasorelaxant Effects.

GPR18 is an orphan GPCR that is activated by the cannabinoid tetrahydrocannabinol (THC). Emerging evidence indicates its involvement in the control of cardiovascular functions, including heart rate, contractility, vascular tone, as well as blood pressure. Therefore, we investigated the effects of selective GPR18 receptor ligands, namely PSB-KD-107 (agonist) and PSB-CB-92 (antagonist), on blood pressure, electrocardiogram (ECG), and vascular dilatation in vitro and in vivo, as well as their anti-oxidative potential in in vitro ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate free radical (DPPH) assays. Our results clearly show that PSB-KD-107 dilates blood vessels. This effect is related to its activation of GPR18 as it can be blocked by the GPR18 antagonist PSB-CB-92. Moreover, our finding confirms the presence of GPR18 in blood vessels. The mechanism of the vasorelaxant activity of PSB-KD-107 is mainly related to endothelial nitric oxide generation; however, we cannot exclude additional nitric oxide-independent mechanisms or a direct influence on K+ channels. PSB-KD-107 may affect blood pressure and heart function after a single administration; however, this effect was no longer observed after repeated administrations once daily for eight days. PSB-KD-107 does not affect platelet aggregation—an important feature considering the safety of its administration. PSB-KD-107 also shows a significant anti-oxidant effect and further studies of its antioxidant activity in vivo are justified.

β‐Hydroxybutyrate (βHOB) Increases Nitric Oxide Synthase Activity in Resistance Arteries from Dahl Salt‐sensitive Rats

The deleterious link between overconsumption of dietary salt and hypertension is unequivocal. This dogma has recently expanded with the report that a high salt diet lowers the circulating levels of the ketone metabolite, beta‐hydroxybutyrate (βHOB), in Dahl salt‐sensitive rats (Dahl S‐HS). While stimulators of ketogenesis are well known for improving cardiovascular health (e.g., exercise and caloric restriction), less is known about negative‐regulation of ketone body metabolism and how it may impact hypertension and vascular aging. Therefore, we hypothesized that raising the circulating levels of βHOB would improve endothelium‐dependent relaxation in mesenteric resistance arteries (MRA) of Dahl S‐HS. To test this hypothesis, 1,3‐butanediol (20% v/v), which is a precursor of βHOB, was supplemented in the drinking water of 5 week old, male Dahl S‐HS for 8 weeks. Subsequently MRA function was analyzed using wire myographs. Although relaxation to endothelium‐dependent vasodilator acetylcholine (ACh) was not different [%Relaxation, Dahl S‐low salt (LS): 40±11 vs. Dahl S‐HS: 52±11 vs. Dahl S‐HS+βHOB: 44±11, p>0.05], the mechanism of relaxation was different between the groups. Specifically, while nitric oxide synthase inhibitor (L‐NAME; 100 μM) completely prevented relaxation in Dahl S‐LS and Dahl S‐HS+βHOB groups, MRA from Dahl S‐HS still partially relaxed in the presence of L‐NAME (%Relaxation, Dahl S‐LS: 1±4 vs. Dahl S‐HS: 33±5 vs. Dahl S‐HS+βHOB: 6±4, p<0.05; Figure 1A–C). These data suggest that MRA from Dahl S‐HS relax through nitric oxide‐independent mechanisms and that βHOB treatment restores nitric oxide synthase activity, despite consumption of a high salt diet. To test whether 1,3‐butanediol treatment had direct effects on arterial function, concentrations‐response curves were performed to βHOB and 1,3‐butanediol in MRA from naïve 5 week old, male Dahl S‐LS rats. We observed that βHOB and 1,3‐butanediol caused triphasic‐quadriphasic responses after pre‐contraction to phenylephrine (PE; Figure 2A–B). Specifically, we observed that low concentrations (<3 nM) of βHOB caused pronounced vasodilation and this was inhibited by both L‐NAME and tetraethylammonium (TEA; potassium channel inhibitor; both, p<0.05; Figure 3A–B). Intermediate concentrations of βHOB (3 nM–100 nM) caused contraction and this tended to be inhibited by indomethacin (cyclooxygenase inhibitor; p=0.08; Figure 3C). Finally, high concentrations (100 nM–30 μM) of βHOB caused relaxation again and this was predominately mediated by nitric oxide (p<0.05; Figure 3A). Overall, these data suggest that βHOB stimulates the production of endothelium‐derived factors and low concentrations of βHOB could be a novel therapy against hypertension‐associated vascular aging by increasing nitric oxide synthesis.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (K99GM118885 and R01HL143082).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nanoliposomal Buparvaquone Immunomodulates Leishmania infantum-Infected Macrophages and Is Highly Effective in a Murine Model.

Visceral leishmaniasis is a fatal parasitic neglected disease affecting 1.5 million people worldwide. Based on a drug repositioning approach, the aim of this work was to investigate the in vitro immunomodulatory potential of buparvaquone (BPQ) and to establish a safe regimen to evaluate the in vivo efficacy of BPQ entrapped by negatively charged nanoliposomes (BPQ-LP) in Leishmania infantum-infected hamsters. Small-angle X-ray scattering, dynamic light scattering, and the ζ-potential were applied in order to study the influence of BPQ on the liposome structure. Our data revealed that BPQ was located in the polar-apolar interface, snorkeling the polar region, and protected against aggregation inside the lipophilic region. The presence of BPQ also decreased the Z-average hydrodynamic diameter and increased the surface charge. Compared to intravenous and intramuscular administration, a subcutaneous route was a more effective route for BPQ-LP; at 0.4 mg/kg, BPQ-LP reduced infection in the spleen and liver by 98 and 96%, respectively. Treatment for 5 days resulted in limited efficacy, but 10 days of treatment resulted in an efficacy similar to that of a 15-day regimen. The nanoliposomal drug was highly effective, with a mean 50% effective dose of 0.25 mg/kg, reducing the parasite load in bone marrow by 80%, as detected using quantitative PCR analysis. In addition, flow cytometry studies showed that BPQ upregulated cytokines as tumor necrosis factor, monocyte chemoattractant protein 1, interleukin-10 (IL-10), and IL-6 in Leishmania-infected macrophages, eliminating the parasites via a nitric oxide-independent mechanism. This new formulation proved to be a safe and effective treatment for murine leishmaniasis that could be a useful candidate against visceral leishmaniasis.

Phagocytosis of hemozoin by RAW 264.7 cells, but not THP-1 cells, promotes infection by Leishmania donovani with a nitric oxide-independent mechanism

During its intra-erythrocytic development, the malaria parasite Plasmodium falciparum synthesizes insoluble hemozoin (HZ) crystals that are released into the circulation upon rupture of parasitized red blood cells, and rapidly phagocytized by host mononuclear cells. Here, HZ persists undigested, causing functional impairment and possibly leading to increased host susceptibility to secondary infections. In patients with malaria and visceral leishmaniasis (VL) co-infections, HZ-loaded macrophages are likely to co-harbor Leishmania donovani parasites, but whether this might influence the course of the Leishmania infection is unknown. In this study, L. donovani amastigote growth was monitored in mouse RAW 264.7 macrophages and PMA-differentiated THP-1 cells previously exposed to increasing amounts of HZ or its synthetic analogue β-hematin (BH). Latex beads were used as a phagocytic control. Data demonstrate that phagocytosis of HZ and BH by RAW 264.7 cells promoted infection therein by L. donovani parasites in a dose-dependent fashion. Similar results were not observed when using THP-1 cells, despite a clear persistence of undigested heme up to 48h after phagocytosis.Conditioning with lipopolysaccharide (LPS)/interferon (IFN)-γ prior to Leishmania infection triggered the release in RAW 264.7 cells of nitric oxide (NO), a highly leishmanicidal metabolite. However, neither HZ nor BH pre-ingestion were able to inhibit NO production following stimulation with LPS/IFN-γ, suggesting that the HZ- and BH-promoting effect on L. donovani infection occurred with an NO-independent mechanism. In conclusion, these preliminary findings highlight a possible detrimental effect of HZ on the course of VL, warranting further investigation into the clinical relevance of the current models.

Mineralocorticoid Receptor Antagonism Treats Obesity-Associated Cardiac Diastolic Dysfunction

Patients with obesity and diabetes mellitus exhibit a high prevalence of cardiac diastolic dysfunction (DD), an independent predictor of cardiovascular events for which no evidence-based treatment exists. In light of renin-angiotensin-aldosterone system activation in obesity and the cardioprotective action of mineralocorticoid receptor (MR) antagonists in systolic heart failure, we examined the hypothesis that MR blockade with a blood pressure-independent low-dose spironolactone (LSp) would treat obesity-associated DD in the Zucker obese (ZO) rat. Treatment of ZO rats exhibiting established DD with LSp normalized cardiac diastolic function, assessed by echocardiography. This was associated with reduced cardiac fibrosis, but not reduced hypertrophy, and restoration of endothelium-dependent vasodilation of isolated coronary arterioles via a nitric oxide-independent mechanism. Further mechanistic studies revealed that LSp reduced cardiac oxidative stress and improved endothelial insulin signaling, with no change in arteriolar stiffness. Infusion of Sprague-Dawley rats with the MR agonist aldosterone reproduced the DD noted in ZO rats. In addition, improved cardiac function in ZO-LSp rats was associated with attenuated systemic and adipose inflammation and an anti-inflammatory shift in cardiac immune cell mRNAs. Specifically, LSp increased cardiac markers of alternatively activated macrophages and regulatory T cells. ZO-LSp rats had unchanged blood pressure, serum potassium, systemic insulin sensitivity, or obesity-associated kidney injury, assessed by proteinuria. Taken together, these data demonstrate that MR antagonism effectively treats established obesity-related DD via blood pressure-independent mechanisms. These findings help identify a particular population with DD that might benefit from MR antagonist therapy, specifically patients with obesity and insulin resistance.

Correction: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

Correction: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

Macrophage control of phagocytosed mycobacteria is increased by factors secreted by alveolar epithelial cells through nitric oxide independent mechanisms.

Tissue-resident macrophages are heterogeneous with tissue-specific and niche-specific functions. Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo. We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC) can influence the functionality of resident pulmonary macrophages (PuM). We have previously reported that factors secreted by AEC increase control of intracellular growth of BCG in macrophages. In the current study, we also aimed to investigate possible mechanisms by which AEC-derived factors increase intracellular control of BCG in both primary murine interstitial macrophages, and bone marrow-derived macrophages and characterize further the effect of these factors on macrophage differentiation. We show that; a) in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b) the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS)1; c) AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS), secretion of nitric oxide (NO), or IL-12, d) in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production. Collectively, our data show that PuM were restricted in inflammatory responses mediated by IFN-γ through SOCS1 and that factors secreted by AEC- enhanced the microbicidal capacities of macrophages by iNOS independent mechanisms.

Melatonin improves cerebrovascular function and decreases oxidative stress in chronically hypoxic lambs.

Chronic hypoxia during gestation and delivery results in oxidative stress and cerebrovascular dysfunction in the neonate. We assessed whether melatonin, a potent antioxidant and potential vasodilator, improves the cerebral vascular function in chronically hypoxic neonatal lambs gestated and born in the highlands (3600 m). Six lambs received melatonin (1 mg/kg per day oral) and six received vehicle, once a day for 8 days. During treatment, biometry and hemodynamic variables were recorded. After treatment, lambs were submitted to a graded FiO2 protocol to assess cardiovascular responses to oxygenation changes. At 12 days old, middle cerebral arteries (MCA) were collected for vascular reactivity, morphostructural, and immunostaining evaluation. Melatonin increased fractional growth at the beginning and improved carotid blood flow at all arterial PO2 levels by the end of the treatment (P < 0.05). Further, melatonin treatment improved vascular responses to potassium, serotonin, methacholine, and melatonin itself (P < 0.05). In addition, melatonin enhanced the endothelial response via nitric oxide-independent mechanisms in isolated arteries (162 ± 26 versus 266 ± 34 AUC, P < 0.05). Finally, nitrotyrosine staining as an oxidative stress marker decreased in the MCA media layer of melatonin-treated animals (0.01357 ± 0.00089 versus 0.00837 ± 0.00164 pixels/μm2 , P < 0.05). All the melatonin-induced changes were associated with no systemic cardiovascular alterations in vivo. In conclusion, oral treatment with melatonin modulates cerebral vascular function, resulting in a better cerebral perfusion and reduced oxidative stress in the neonatal period in chronically hypoxic lambs. Melatonin is a potential therapeutic agent for treating cerebrovascular dysfunction associated with oxidative stress and developmental hypoxia in neonates.

Interleukin 1β attenuates vascular α1 adrenergic receptors expression following lipopolysaccharide-induced endotoxemia in rabbits

Studies have shown that interleukin 1β (IL-1β) participates in the down-regulation of vascular reactivity via both nitric oxide-dependent and nitric oxide-independent mechanisms during shock. However, the precise mechanisms of nitric oxide-independent pathway remain to be established. The effect of IL-1β on the expression of α1 adrenergic receptors (α1AR) and the relationship with Janus kinase 2-signal transducer and activator of transcription 3 (JAK2-STAT3) pathway were observed using a rabbit model of lipopolysaccharide (LPS)-induced endotoxemia and superior mesenteric arteries (SMAs) in vivo and in vitro, respectively. The vascular reactivity of SMAs to α1AR agonist (phenylephrine) displayed a biphasic change after LPS (significantly increased at 0.5 hour following LPS and then markedly decreased after 2 hours), the α1A, α1B and α1DAR messenger RNA (mRNA) and protein expression seemed a time-dependent decrease following LPS administration, α1A and α1DAR decreased more obviously than α1BAR. IL-1ra (4 µg/mL) partly reversed LPS-induced the decrease of vascular reactivity and down-regulation of α1AR expression. In vitro incubation with IL-1β (12.5-50 ng/mL) significantly decreased the vascular reactivity of SMA to phenylephrine and the expression of α1AR mRNA and protein and elevated the DNA binding ability of STAT3. AG490 (10 µmol/L), an inhibitor of JAK2, partly reversed the IL-1β-induced down-regulation of vascular reactivity and α1AR mRNA and protein expression and suppressed the DNA binding ability of STAT3. IL-1β participates in the regulation of vascular hyporeactivity following endotoxemia in rabbit. The mechanism is related to the down-regulation of α1AR expression through activating the JAK2-STAT3 pathway.

Sustained activation of AMPK ameliorates age-associated vascular endothelial dysfunction via a nitric oxide-independent mechanism

Exercise restores endothelium-dependent dilation (EDD) in old mice by reducing oxidative stress and increasing nitric oxide (NO) bioavailability. Adenosine monophosphate protein kinase (AMPK) activation mimics some effects of exercise. Old (28–30 months) B6D2F1 mice had reduced arterial AMPK expression and superoxide-mediated suppression of EDD vs. young (3–6 months) controls. Pharmacological activation of AMPK by aminoimidazole carboxamide ribonucleotide (AICAR) for 2 weeks increased arterial AMPK and reversed this superoxide-induced impairment of EDD. The improvement in EDD was independent of NO or prostaglandin signaling, suggesting enhanced endothelium-dependent hyperpolarizing factor-related dilation. AMPK activation may represent a novel therapy for treating age-associated vascular dysfunction.

DHA-rich fish oil reverses the detrimental effects of saturated fatty acids on postprandial vascular reactivity

Experimental elevation of nonesterified fatty acids (NEFAs) impairs endothelial function, but the effect of NEFA composition is unknown. The objective was to test the effect of acute elevation of NEFAs enriched with either saturated fatty acids (SFAs) or SFAs with long-chain (LC) n-3 (omega-3) PUFAs on vascular function measured via flow-mediated dilatation (FMD), laser Doppler iontophoresis (LDI), and digital volume pulse (DVP). In 59 subjects (30 men and 29 women), repeated oral fat feeding of either palm stearin (SFA) or palm stearin with DHA-rich fish oil (SFA + LC n-3 PUFA) was performed on 2 separate occasions with continuous heparin infusion to elevate NEFAs for a duration of 60 to 240 min. Vascular function was measured at baseline and at the end of NEFA elevation; venous blood was collected for measurement of lipids and circulating markers of endothelial function. NEFA elevation during consumption of the SFA-rich drinks was associated with a marked impairment of FMD, whereas consumption of SFAs + LC n-3 PUFAs improved FMD response, with a mean (±SEM) difference of 2.06 ± 0.29% (P < 0.001). Positive correlations were found with percentage weight of LC n-3 PUFAs in circulating NEFAs and change in FMD response [Spearman's rho (r(s)) = 0.460, P < 0.001]. LDI measures increased during both treatments (P ≤ 0.026), and there was no change in DVP indexes. The composition of NEFAs can acutely affect FMD. The beneficial effect of LC n-3 PUFAs on postprandial vascular function warrants further investigation but may be mediated by nitric oxide-independent mechanisms. This trial is registered at clinicaltrials.gov as NCT01351324.

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300 bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen, blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia coli exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection.

N‐arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium‐activated potassium channels

N-arachidonoyl glycine (NAGly) is an endogenous lipid that is structurally similar to the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). While NAGly does not activate cannabinoid receptors, it exerts cannabimimetic effects in pain regulation. Here, we have determined if NAGly, like anandamide, modulates vascular tone. In rat isolated small mesenteric arteries, the relaxant responses to NAGly were characterized. Effects of N-arachidonoyl serine and N-arachidonoyl gamma-aminobutyric acid were also examined. In endothelium-intact arteries, NAGly-induced relaxation (pEC(50%)= 5.7 +/- 0.2; relaxation at 30 microM = 98 +/- 1%) was attenuated by l-NAME (a nitric oxide synthase inhibitor) or iberiotoxin [selective blocker of large conductance Ca(2+)-activated K(+) channels (BK(Ca))], and abolished by high extracellular K(+) concentration. Endothelial removal reduced the potency of NAGly, and the resultant relaxation was inhibited by iberiotoxin, but not l-NAME. NAGly responses were sensitive to the novel cannabinoid receptor antagonist O-1918 independently of endothelial integrity, whereas pertussis toxin, which uncouples G(i/o) proteins, attenuated NAGly relaxation only in endothelium-intact arteries. Treatments with antagonists for CB(1), CB(2) and TRPV1 receptors, or inhibitors of fatty acid amide hydrolase and COX had no effect. The two other arachidonoyl amino acids also induced iberiotoxin- and L-NAME-sensitive relaxations. NAGly acts as a vasorelaxant predominantly via activation of BK(Ca) in rat small mesenteric arteries. We suggest that NAGly activates an unknown G(i/o)-coupled receptor, stimulating endothelial release of nitric oxide which in turn activates BK(Ca) in the smooth muscle. In addition, NAGly might also activate BK(Ca) through G(i/o)- and nitric oxide-independent mechanisms.

Increased blood flow induces oxidative stress through an endothelium- and nitric oxide-independent mechanism

Bradykinin, a potent vasodilator, stimulates the formation of reactive oxygen species and F2-isoprostanes in vitro. The effect of bradykinin on oxidative stress in humans is not known. This study tested the hypothesis that bradykinin induces oxidative stress through a nitric oxide (NO)-dependent mechanism in the human vasculature. We measured forearm blood flow (FBF) and net F2-isoprostane release in response to intraarterial bradykinin (50–200ng/min), nitroprusside (1.6–6.4µg/min), or diltiazem (3.6–14.4µg/min) in the absence and presence of the NO synthase inhibitor Nω-monomethyl-L-arginine (L-NMMA) in normotensive and hypertensive subjects pretreated with aspirin. L-NMMA significantly decreased basal FBF and basal net F2-isoprostane release (from 28.7±5.2 to 13.4±3.5pg/min/100ml, P=0.01) in all subjects. Bradykinin caused a significant increase in FBF and net F2-isoprostane release in both normotensive and hypertensive subjects. During NO synthase inhibition, bradykinin significantly increased net F2-isoprostane release in both groups (P=0.001) and there was no effect of L-NMMA on bradykinin-stimulated F2-isoprostane release (P=0.46). Nitroprusside also significantly increased net F2-isoprostane release in hypertensive subjects (P=0.01) and this response was not affected by L-NMMA (P=0.50). Diltiazem increased FBF as well as net F2-isoprostane release (from 44.5±12.5 to 61.2±14.7pg/min/100ml at the highest dose, P=0.05). Increasing blood flow induces oxidative stress through a NO- and endothelium-independent mechanism.

Effect Of Elevated Local Temperature On Cutaneous Vasoconstrictor Responsiveness In Humans

Cutaneous vascular conductance (CVC) increases in response to local skin heating. While attenuation of vasoconstrictor responsiveness due to local heating has been demonstrated, the mechanism(s) responsible for this attenuation remain unclear. Nitric oxide has been shown to at least partially contribute to this response, but other mechanisms also may be involved. PURPOSE: To test the hypothesis that local heating diminishes cutaneous vasoconstrictor responsiveness through a nitric oxide independent mechanism by altering post-synaptic reactivity to norepinephrine. A follow-up protocol tested the hypothesis that local heating attenuates the pre-synaptic release of neurotransmitters that cause vasoconstriction, also via non-nitric oxide mechanisms. METHODS: Protocol I: in 8 subjects, CVC was assessed while increasing doses of norepinephrine (via intradermal microdialysis) were administered at adjacent sites separately heated to 34 and 40 °C. Protocol II: in an identical but separate protocol, in 7 subjects, CVC was assessed while increasing doses of tyramine, which causes release of neurotransmitters from adrenergic nerves, were administered. At each site for both protocols, nitric oxide synthesis was inhibited (via microdialysis administration of NG-nitro-L-arginine methyl ester) and flow was matched (via microdialysis administration of adenosine), leaving temperature as the only variable different between sites. RESULTS: For both protocols, non-linear regression analysis revealed no difference (P > 0.05) in the effective drug concentration causing 50% of the vasoconstrictor response. Minimum CVC (protocol I, mean ± SD: 6.3 ± 2.0 and 9.0 ± 4.0 %CVCpeak; protocol II: 19.3 ± 9.3 and 20.5 ± 11.9 %CVCpeak at the 34 and 40 °C sites, respectively) was not different between sites. CONCLUSION: Independent of nitric oxide, local skin heating to 40 °C does not attenuate adrenergically-mediated cutaneous vasoconstriction through pre- or post-synaptic mechanisms. Supported by NIH Grants HL61388, HL84072, and GM68865.

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40–CD40L system

The vascular effects of nitrolinoleate (LNO 2), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO 2 was capable to deliver free radical nitric oxide ( ·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO 2 for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO 2 were observed in vivo by intravital microscopy assays. LNO 2 decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO 2 reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO 2 on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO 2 involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO 2 inhibited adhesion molecules expression and promoted ·NO inactivation of the CD40–CD40L system, both important processes of the inflammatory response.

Effect of elevated local temperature on cutaneous vasoconstrictor responsiveness in humans

Cutaneous vascular conductance (CVC) increases in response to local skin heating. Although attenuation of vasoconstrictor responsiveness due to local heating has been demonstrated, the mechanism(s) responsible for this attenuation remains unclear. Nitric oxide has been shown to at least partially contribute to this response, but other mechanisms also may be involved. The purpose of this study was to test the hypothesis that local heating diminishes cutaneous vasoconstrictor responsiveness through a nitric oxide-independent mechanism by altering postsynaptic reactivity to norepinephrine. A follow-up protocol tested the hypothesis that local heating attenuates the presynaptic release of neurotransmitters that cause vasoconstriction, also via non-nitric oxide mechanisms. In protocol I, CVC was assessed in eight subjects during administration of increasing doses of norepinephrine (via intradermal microdialysis) at adjacent sites separately heated to 34 degrees C and 40 degrees C. In protocol II, which was identical to, but separate from, protocol I, CVC was assessed in seven subjects during administration of increasing doses of tyramine, which causes release of neurotransmitters from adrenergic nerves. At each site for both protocols, nitric oxide synthesis was inhibited (via microdialysis administration of N(G)-nitro-l-arginine methyl ester) and flow was matched (via microdialysis administration of adenosine); therefore, temperature was the only variable that differed between the sites. For both protocols, nonlinear regression analysis revealed no difference (P > 0.05) in the effective drug concentration causing 50% of the vasoconstrictor response. Minimum CVC [6.3 +/- 2.0 and 9.0 +/- 4.0% of peak CVC (mean +/- SD) for protocol I and 19.3 +/- 9.3 and 20.5 +/- 11.9% of peak CVC for protocol II at 34 degrees C and 40 degrees C sites, respectively] was not different between sites. Independent of nitric oxide, local skin heating to 40 degrees C does not attenuate adrenergically mediated cutaneous vasoconstriction through pre- or postsynaptic mechanisms.