Enhanced Nitric Oxide Release Research Articles

Dual photoresponsive & water-triggered nitric oxide releasing materials based on rhodium-based metal-organic polyhedra.

The exogenous administration of nitric oxide (NO) is considered a potential therapeutic treatment against a great variety of diseases due to its significant role in multiple physiological functions. Due to the gaseous nature, short lifetime and dose- and tissue-dependent activity of this molecule, the development of new administration procedures is required to control the NO delivery in terms of dosage, timing, and location. In this work, we propose a new molecular material based on robust metal-organic polyhedra (MOPs) for controlled NO release. We select dirhodium paddlewheel complex-based cuboctahedral MOPs (RhMOP), in which NO can chemically coordinate to the open-metal sites at the axial sites of dirhodium paddlewheel moieties. We further prepare amorphous coordination polymer particles (CPPs) by connecting RhMOP with bis(imidazole) linkers at the external axial sites. Both molecular MOPs and polymeric CPPs show relevant NO payloads and the release of NO can be triggered by two different stimuli: light and humidity. We show that imidazole ligands coordinating to the external axial sites of the paddlewheel moieties tune the light-triggered NO release property. We further demonstrate that the size and the extrinsic pores of CPPs are important for enhanced NO release.

Spirulina extract improves age-induced vascular dysfunction

Context Vascular dysfunction is considered a hallmark of ageing that has been associated with altered vasomotor responses, in which nitric oxide (NO) and reactive oxygen species participate. The consumption of Spirulina extracts, with antioxidant properties, increased recently. Objective This study investigates the effect of Spirulina aqueous extract (SAE) on the vascular function of the aorta from aged rats. Materials and methods Aortic segments from aged male Sprague-Dawley rats (20–22 months old) were exposed to SAE (0.1% w/v, for 3 h) to analyse: (i) the vasodilator response induced by acetylcholine (ACh), by the NO donor sodium nitroprusside (SNP), by the carbon monoxide releasing molecule (CORM) and by the KATP channel opener, cromakalim (CK); (ii) the vasoconstrictor response induced by KCl and noradrenaline (NA); (iii) the production of NO and superoxide anion, and (iv) the expression of the p-eNOS and HO-1 proteins. Results Incubation with SAE increased the expression of p-eNOS (1.6-fold) and HO-1 (2.0-fold), enhanced NO release (1.4-fold in basal and 1.9-fold in ACh-stimulated conditions) while decreased the production of superoxide (0.7-fold). SAE also increased the sensitivity (measured as pEC50) to ACh (control: −7.06 ± 0.11; SAE: −8.16 ± 0.21), SNP (control: −7.96 ± 0.16; SAE: −9.11 ± 0.14) and CK (control: −7.05 ± 0.39; SAE: −8.29 ± 0.53), and potentiated the response to KCl (1.3-fold) and to NA (1.7-fold). Conclusion The antioxidant properties of SAE improved the vasomotor responses of aorta from aged rats. These results may support the use of Spirulina as a protection against vascular dysfunction.

Surface-Catalyzed Nitric Oxide Release via a Metal Organic Framework Enhances Antibacterial Surface Effects.

It has been previously demonstrated that metal nanoparticles embedded into polymeric materials doped with nitric oxide (NO) donor compounds can accelerate the release rate of NO for therapeutic applications. Despite the advantages of elevated NO surface flux for eradicating opportunistic bacteria in the initial hours of application, metal nanoparticles can often trigger a secondary biocidal effect through leaching that can lead to unfavorable cytotoxic responses from host cells. Alternatively, copper-based metal organic frameworks (MOFs) have been shown to stabilize Cu2+/1+ via coordination while demonstrating longer-term catalytic performance compared to their salt counterparts. Herein, the practical application of MOFs in NO-releasing polymeric substrates with an embedded NO donor compound was investigated for the first time. By developing composite thermoplastic silicon polycarbonate polyurethane (TSPCU) scaffolds, the catalytic effects achievable via intrapolymeric interactions between an MOF and NO donor compound were investigated using the water-stable copper-based MOF H3[(Cu4Cl)3(BTTri)8-(H2O)12]·72H2O (CuBTTri) and the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). By creating a multifunctional triple-layered composite scaffold with CuBTTri and SNAP, the surface flux of NO from catalyzed SNAP decomposition was found tunable based on the variable weight percent CuBTTri incorporation. The tunable NO surface fluxes were found to elicit different cytotoxic responses in human cell lines, enabling application-specific tailoring. Challenging the TSPCU-NO-MOF composites against 24 h bacterial growth models, the enhanced NO release was found to elicit over 99% reduction in adhered and over 95% reduction in planktonic methicillin-resistant Staphylococcus aureus, with similar results observed for Escherichia coli. These results indicate that the combination of embedded MOFs and NO donors can be used as a highly efficacious tool for the early prevention of biofilm formation on medical devices.

Liriodendrin, ameliorates hypertension by calcium channel blockade and enhancing enos expression in wistar rats

Introduction: Hypertension is found to be the prime cause ofdeath worldwide in spite of a number of available treatmentswhich suggests that there is a need of discovering new leadmolecules that would be more effective to treat cardiovasculardisease (CVD). Liriodendrin, the lignan phytoconstituent possessespotential pharmacological effects. Literature survey suggeststhat liriodendrin could be effective in mitigating hypertensionconsidering its structural similarity with reported cardiovascularprotective drugs. Hence liriodendrin is investigated to reveal itsmechanism of actions to support its antihypertensive property.Methods: Hypertension was induced in male wistar rats withDOCA salt. Hypertensive rats were treated with liriodendrin for4 weeks. Blood pressure, heart rate, body weight, lipid profile,serum nitrite levels, vascular reactivity to various catecholamines,in-vitro calcium channel blocking assays, antioxidantassay, determination of aortic calcium level, endothelial function,expression of eNOS analysis were studied.Result: Liriodendrin was found safe orally up to 2000 mg/kg. Itshowed a significant decrease in heart rate, blood pressure andmean arterial pressure. In-vitro study on the isolated rat aorta revealedthe calcium channel blocking potential of liriodendrin. Vascularreactivity to various catecholamines was normalized. Vascularendothelium was significantly protected by the enhancedrelease of nitric oxide and eNOS expression by the western blottechnique. Oxidative stress was also significantly reduced.Conclusion: Liriodendrin was found to be beneficial in hypertensionas it produced vasorelaxation by blocking calciumchannels, enhancing nitric oxide release, and reducing oxidativestress. Thus, liriodendrin may be useful to relieve hypertensionand cardiovascular complications.

Endogenous NO-releasing Carbon Nanodots for Tumor-specific Gas Therapy

Carbon nanodots based on L-arginine (L-Arg) were developed for enhanced nitric oxide (NO) gas therapy for cancer. The L-Arg-based carbon nanodots (Arg-dots) produced high levels of NO in the tumor environment rich in endogenous H2O2. In vitro cell experiments revealed that the Arg-dots could kill tumor cells (including human breast cancer cell line MCF-7, female gastric cancer cell line BGC-823, male lung cancer cell line A549, and female leukemic cell line K562) but did not affect the activity of normal cells (human normal lung epithelial cell line BEAS-2B). The Arg-dots produced twice the amount of NO for an equivalent amount of L-Arg. Theoretical calculations showed that the carbonization structure of the Arg-dots promoted significantly more electrons toward the guanidinium groups of L-Arg and boosted the adsorption of H2O2 molecules. In vitro and in vivo investigations confirmed that the Arg-dots reduced the multidrug resistance (MDR) effect of the tumor cells (MCF-7/ADR cells) and produced a combined antitumor efficacy with traditional chemotherapeutic drugs (adriamycin [ADR]). The fluorescence property (quantum yield, 6.88%) allows the Arg-dots to be used as a suitable fluorescent probe for fluorescence imaging of tumor cells. The ultra-small size of the Arg-dots (diameter: ca. 2.5 nm) enables them not only to penetrate deep tumors and provide enhanced antitumor activity but also to be removed through kidney filtration and have a renal clearance property. Statement of significanceNitric oxide (NO), which serves as a biological messenger, can be used in gas therapy for cancer. The development of a safe and efficient NO cancer therapy is, however, challenging because of the low NO release amount and poor tumor specificity of most NO donors. Many efforts have been made to overcome these drawbacks, but solving both these limitations through a single approach has been seldom achieved. In the present work, carbon nanodots (Arg-dots) from L-arginine were used for gas therapy of cancer. The Arg-dots produced NO in the H2O2-rich tumor environment. Theoretical calculations were consistent with the mechanism of enhanced NO release amount. The Arg-dots also reduced the multidrug resistance effect in cancer chemotherapy. In vivo and in vitro toxicity assessments confirmed that the Arg-dots have excellent biosafety.

TLR2- and TLR3-activated microglia induce different levels of neuronal network dysfunction in a context-dependent manner

Recognition of pathogen- or damage-associated molecular patterns (PAMPs, DAMPs) by innate Toll-like receptors (TLRs) is central to the activation of microglia (brain macrophages) in many CNS diseases. Notably, TLR-mediated microglial activation is complex and modulated by additional exogenous and endogenous immunological signals. The impact of different microglial reactive phenotypes on electrical activity and neurotransmission is widely unknown, however. We explored the effects of TLR ligands on microglia and neuronal network function in rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortical tissue lacking adaptive immunity. Single exposure of slice cultures to TLR2 or TLR3 ligands [PGN, poly(I:C)] for 2–3 days induced moderate microglial activation featuring IL-6 and TNF-α release and only mild alterations of fast neuronal gamma band oscillations (30–70 Hz) that are fundamental to higher cognitive functions, such as perception, memory and behavior. Paired exposure to TLR3/TLR2 or TLR3/TLR4 ligands (LPS) induced nitric oxide (NO) release, enhanced TNF-α release, and associated with advanced network dysfunction, including slowing to the beta frequency band (12–30 Hz) and neural bursts (hyperexcitability). Paired exposure to a TLR ligand and the leukocyte cytokine IFN-γ enhanced NO release and associated with severe network dysfunction, albeit sensitive parvalbumin- and somatostatin-positive inhibitory interneurons were preserved. Notably, the neuronal disturbance was prevented by either microglial depletion or pharmacological inhibition of oxidant-producing enzymes, inducible NO synthase (iNOS) and NADPH oxidase. In conclusion, TLR-activated microglia can induce different levels of neuronal network dysfunction, in which severe dysfunction is mainly caused by reactive oxygen and nitrogen species rather than proinflammatory cytokines. Our findings provide a mechanistic insight into microglial activation and functional neuronal network impairment, with relevance to neuroinflammation and neurodegeneration observed in, e.g., meningoencephalitis, multiple sclerosis and Alzheimer’s disease.

The Effects of Novel Myristoylated PKC Epsilon Peptide Modulators on Real‐Time Nitric Oxide Release in Human Umbilical Vein Endothelial Cells

Endothelial dysfunction is a major consequence of ischemia‐reperfusion (I/R) injury, such as during the re‐establishment of blood flow after endovascular thrombus removal. It is characterized by limited nitric oxide (NO) bioavailability and production of superoxide (SO) instead of NO, in part from uncoupled endothelial NO synthase (eNOS) activity when the dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4) ratio is elevated during reperfusion following prolonged ischemia. The protein kinase C epsilon (PKCɛ) isoform is known to participate in pre‐conditioning (PKCɛ activation treatment prior to ischemia) and post‐conditioning (PKCɛ inhibition treatment during reperfusion) to attenuate rat myocardial I/R injury. Normally following diacylglycerol‐dependent activation, PKCɛ binds to isoform‐specific receptors for activated C‐kinase (RACK) and translocates from the cytosol to the cell membrane to phosphorylate its targets, such as eNOS at Ser‐1177 to augment NO release when the BH2 to BH4 ratio is reduced to promote coupled eNOS activity that is normally seen prior to prolonged ischemia. Cell permeable myristic acid conjugated PKCɛ peptide activator (myr‐HDAPIGYD; myr‐PKCɛ+) and inhibitor (N‐myr‐EAVSLKPT; myr‐PKCɛ‐) has been shown to increase and decrease NO release, respectively, in rat aortic tissue by regulating PKCɛ translocation to eNOS. However, modulation of PKCɛ‐mediated NO release with these peptides in human cells remains undetermined. We hypothesize that myr‐PKCɛ+ will augment and myr‐PKCɛ‐ will attenuate NO release in cultured human umbilical vein endothelial cells (HUVECs). Real‐time HUVEC NO release (pmol) was measured using a calibrated NO electrode following administration of myr‐PKCɛ+ or myr‐PKCɛ‐ ± acetylcholine (Ach; positive control). Data were analyzed by ANOVA using Fishers post‐hoc analysis. Basal NO levels (66±6; n=35) were determined as the pmol difference between cell‐populated (106 cells/well) and reagent only wells. Control Ach (10 μM) enhanced NO release (102±7; n=37), myr‐PKCɛ+ (10 μM) enhanced NO release with Ach (107±16; n=8) and without Ach (107±13; n=7), and myr‐PKCɛ‐ (10 μM) attenuated NO release with Ach (37±9; n=13) and without Ach (7±29; n=9) compared to basal levels (all p<0.05). Results suggest that myr‐PKCɛ +/− effects on NO release is translational across mammalian species, presumably through activation and inhibition of PKCɛ‐mediated phosphorylation of eNOS. Thus, therapeutic intervention that targets inhibition of PKCɛ to reduce uncoupled eNOS activity during reperfusion may yield protective effects via attenuating the extent of I/R injury seen in clinical myocardial infarction, coronary bypass, coronary angioplasty, and organ transplantation.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current research license is supported by Young Therapeutics, LLC.

Novel nitric oxide-generating platform using manuka honey as an anti-biofilm strategy in chronic rhinosinusitis.

Bacterial biofilms are implicated in the pathogenesis of chronic rhinosinusitis. Nitric oxide (NO) is a key immune effector with potent antimicrobial effects, but a short half-life limits achievement of therapeutic concentrations. We hypothesized that manuka honey (MH) could induce sustained reduction of nitrite to NO causing biofilm disruption and that this effect would be enhanced with the addition of a NO-releasing microparticle. Porous organosilica microparticles containing nitrosylated thiol groups were formulated (SNO-MP). MH was combined with serial dilutions of nitrite. NO release was evaluated using a NO analyzer. The susceptibility of 2 strains of Pseudomonas aeruginosa biofilms to these NO-releasing platforms was evaluated using confocal microscopy. Cell viability and biofilm volume were quantified. Statistical analysis was performed using the Mann-Whitney U test with SPSS software. MH with nitrite generated a linear increase in NO formation. SNO-MP induced a bolus release of NO within 5 minutes, followed by a sustained plateau phase. MH with nitrite combined with SNO-MP enhanced NO release during the plateau phase. MH with nitrite reduced biofilm live cells and volume by 88.5% to 96.9% and 95.1% to 95.6%, respectively, vs control (p < 0.0001). SNO-MP reduced live cells and volume by 61.0% to 98.5% and 74.7% to 85.7%, respectively, vs control (p < 0.0001). MH with nitrite combined with SNO-MP nearly eradicated biofilm, with a 98.3% to 99.8% (log 1.8-2.6) reduction in viability and a 91.4% to 97.7% decrease in volume (p < 0.0001 vs control). A novel platform that generates NO using MH and nitrite produces a potent anti-biofilm effect, which can be further enhanced with the addition of SNO-MP.

Impact of ginsenoside-Rg3 on catecholamine secretion in the perfused model of the rat adrenal medullae

Background: The present study was the first attempt to explore the characteristics of ginsenoside-Rg3 (Rg3) on release of catecholamines (CA) in the perfused rat adrenal medullae and also to verify the underlying action mechanism. Materials and Methods: The adrenal medulla was separated by some modification of the previous method and perfused with Krebs solution. CA was assayed directly by the fluorometry. Results: Rg3 reduced acetylcholine (ACh)-produced CA release in a dose- and time-dependent manner. Rg3 time-dependently depressed CA release produced by 3-(m-chloro-phenyl-carbamoyl-oxy)-2-butynyltrimethyl ammonium chloride (McN-A-343), 1,1-dimethyl-4-phenyl piperazinium iodide, and angiotensin II. In the presence of Rg3, the CA release produced by high K+, veratridine, cyclopiazonic acid, and methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4- (2-trifluoromethyl-phenyl)-pyridine-5-carboxylate (Bay-K-8644) was also markedly suppressed. However, during the simultaneous perfusion of Rg3 and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), the release of CA produced by ACh, angiotensin II, Bay-K-8644, and veratridine was restored closely to the level of each control, in contrast to that of Rg3-treatment alone. The nitric oxide (NO) release was significantly elevated by Rg3-treatment. Furthermore, in the coexistence of Rg3 and fimasartan, ACh-produced CA release was more significantly reduced as compared to that of fimasartan-treatment alone. Conclusions: We present the first evidence that Rg3 markedly depresses the CA secretion produced by activation of neuronal cholinergic and angiotensinergic receptors. Rg3-produced inhibition appears to be evoked not only by blocking the inflow of Na+ and Ca2+ into adrenomedullary cells but also by preventing the Ca2+ release from intracellular storage, partly through enhancement of NO release by NO synthase activation. Coadministration of Rg3 and fimasartan may be clinically beneficial for the treatment of cardiovascular diseases. Abbreviations used: Rg3: Ginsenoside-Rg3; VDCC: L-type voltage-dependent Ca2+ channels; Bay–K-8644: Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl-phenyl)-pyridine-5-carboxylate; ACh: Acetylcholine; McN-A-343: 3-(m-chloro-phenyl-carbamoyl-oxy)-2-butynyltrimethyl ammonium; DMPP: 1.1-dimethyl-4-phenyl piperazinium; NO: Nitric oxide; L-NAME: Nω-nitro-l-arginine methyl ester; nNOS: Neuronal NO synthase.

277 - Encapsulation of tDodSNO generates a nitric oxide releasing nanoparticle

The development of drugs for the controlled delivery of therapeutic nitric oxide (NO) is an ongoing challenge. Current options include organonitrates and nitroprusside which have severe limitations to their use, with organonitrates inducing tolerance and nitroprusside releasing toxic cyanide ions. An alternative is the s-nitrosothiol drug class which breaks down to release NO; however, this NO release is normally too rapid to be therapeutically useful. The molecule tDodSNO has recently been developed as a stable s-nitrosothiol which is resistant to metabolic and thermal degradation. Its key property is that it can be selectively degraded by light, with photoactivation causing NO release. To render this s-nitrosothiol suitable for in vivo applications we report encapsulating tDodSNO within a nanoparticle consisting of a co-polymer of styrene and maleic acid (SMA). The resulting SMA-tDodSNO was fully water soluble, and the nanoparticle also further protected tDodSNO from degradation by glutathione, with a half-life increase of 24 hours. Encapsulation did not affect tDodSNO’s NO release profile, with similar NO levels (0-2 µM) recorded over a range of photoactivation levels for both tDodSNO and SMA-tDodSNO. Moving into cardiovascular activity assays, photoactivation of SMA-tDodSNO allowed controllable vasodilation of rat aortic rings, with vascular tone proportional to photoactivation. Additionally, in rat mesenteric beds, photoactivated SMA-tDodSNO induced selective hyperpermeability to macromolecules. In comparison to nitroprusside and the s-nitrosothiols GSNO and SNAP, without photoactivation SMA-tDodSNO induced vasodilation at a 40 fold higher EC50. However, upon photoactivation, SMA-tDodSNO was the only molecule to display enhanced NO release, with its EC50 decreasing to GSNO levels. This EC50 shift demonstrated that SMA-tDodSNO can be used to selectively induce NO dependent signalling by photoactivation. Future development of SMA-tDodSNO is therefore predicted to find applications for treating cardiovascular disease, as well as for enhancing the delivery of macromolecules, including antibodies and nanoconstructs, to target tissue.

Metformin treatment decreases nitroxidative stress, restores nitric oxide bioavailability and endothelial function beyond glucose control

Reduction of nitric oxide (NO), a potent vasodilator, and an increase in cytotoxic peroxynitrite (ONOO−) may be associated with the uncoupling of NO synthase (eNOS) and endothelial cell (EC) dysfunction. In addition to its effect on glucose control, metformin, may also directly benefit in the restoration of the function of eNOS and EC. Obese Zucker rats were administered vehicle or 300 mg/kg/day metformin for 4 weeks. NO concentration [NO] and ONOO− concentration [ONOO−] were measured in aortic and glomerular endothelial cells from Zucker rats in vitro. Compared with controls, aortic and glomerular endothelial [NO] was reduced by 32% and 41%, while [ONOO−] release increased 79% and 69%, respectively. Metformin treatment increased aortic and glomerular endothelial [NO] by 37% and 57%, respectively, while decreasing [ONOO−] by 32% and 34%, compared with vehicle-treated animals. Treatment with metformin significantly restored the balance in the [NO]/[ONOO−] ratio with 101% and 138% increase for aortic and glomerular endothelial cells, respectively. Fasting glucose levels were not significantly changed. These findings indicate that metformin therapy has a direct and beneficial effect on arterial and renal EC function in obese rats, including enhanced NO release and reduced nitroxidative stress, beyond any effects on fasting glucose levels.

Enhancement of nitric oxide release and hemocompatibility by surface chirality of D-tartaric acid grafting

Nitric Oxide (NO) generation from endogenous NO-donors catalyzed by diselenide modified biomaterials has been reported. Here we reported surface chirality by L-tartaric acid and D-tartaric acid grafting on the outermost showed a significant impact on diselenide modified biomaterials, which modulated protein adsorption, NO release and anti-platelet adhesion properties. D-tartaric acid grafted surface showed more blood protein adsorption than that of L-surfaces by QCM analysis, however, ELISA analysis disclosed less fibrinogen denatured on the D surfaces. Due to the surface ratio of selenium decreasing, NO release catalyzed by L-tartaric acid grafting on the outermost significantly decreased in comparison to that of only selenocystamine immobilized surfaces. While NO release catalyzed by D-tartaric acid grafting on the outermost didn’t decrease and was similar with that of selenocystamine immobilized surfaces. Surface chirality combined with NO release had synergetic effects on platelet adhesion, and it showed the lowest number of platelets adhered on the D-tartaric acid grafted surfaces. Thus surface chirality from D-tartaric acid grafting enhanced hemocompatibility of the surface in this study. Our work provides new insights into engineering novel blood contacting biomaterials by taking into account surface chirality.

A positive feedback loop between nitric oxide and amyloid β (1-42) accelerates mitochondrial damage in human lens epithelial cells.

We have reported that excessive nitric oxide (NO), like other reactive oxygen species (ROS), causes a decrease in cytochrome c oxidase (CCO) activity and ATP levels (mitochondrial damage) resulting in lens opacity. In addition, previous reports have shown that oxidative stress caused by ROS enhances amyloid β (Aβ) production in mammalian lenses, and that Aβ1-42 stimulates inducible nitric oxide synthase (iNOS) promoter activity. Based on these reports, we investigated the relationship between NO and Aβ1-42 production in human lens epithelial (HLE) cells. iNOS was induced by the co-incubation of HLE cells with 1000 IU interferon-γ (IFN-γ) and 100ng/ml lipopolysaccharide (LPS) for 48h. This led to enhanced NO release, an increase in the gene expression levels of proteins related to Aβ production, and the cellular accumulation of Aβ1-42. Moreover, both aminoguanidine (AG, a selective inhibitor of iNOS) and diethyldithiocarbamate (DDC, a nuclear factor-kappa B (NFκB) inhibitor) attenuated these changes in IFN-γ and LPS stimulated HLE cells. Based on our finding that Aβ1-42 accumulation is induced by co-incubation of HLE cells with both IFN-γ and LPS, we prepared a HLE cell model with Aβ1-42 accumulation (Aβ-accumulated-HLE cell model) by pre-stimulating cells with IFN-γ and LPS for 48h. Aβ1-42 accumulation caused NO production via iNOS, resulting in an enhancement in the mRNA levels for enzymes necessary for the proteolysis of amyloid precursor protein (APP) to Aβ in HLE cells. In addition, excessive NO produced in response to Aβ1-42 accumulation led to a decrease in CCO activity and ATP levels. Taken together, we hypothesize that excessive NO production in the lens epithelium enhances Aβ1-42 production, and that this enhancement accelerates NO release. The enhancement in NO production in the lens epithelium based on positive feedback (NO-Aβ positive feedback loop, a vicious cycle) may promote the onset of cataracts (lens opacification) via the decrease in CCO activity and ATP levels. These findings provide significant information that can be used to design further studies aimed at developing anti-cataract drugs.

Chirality-mediated enhancement of nitric oxide release and regulation of endothelial cells behaviors by cystine immobilization on Ti–O films

NO release inducing by regulated by surface chirality have significant effects on endothelial cells.

Topological control of nitric oxide secretion by tantalum oxide nanodot arrays.

BackgroundNitric oxide (NO) plays a very important role in the cardiovascular system as a major secondary messenger in signaling pathway. Its concentration regulates most of the important physiological indexes including the systemic blood pressure, blood flow, regional vascular tone and other cardiac functions. The effect of nanotopography on the NO secretion in cardiomyocytes has not been elucidated before. In this study, we report how the nanotopography can modulate the secretion profile of NO and attempt to elucidate the genetic pathways responsible for the same by using Tantalum Oxide nanodot arrays ranging from 10 to 200 nm. A series of nanodot arrays were fabricated with dot diameter ranging from 10 to 200 nm. Temporal NO release of cardiomyocytes was quantified when grown on different surfaces. Quantitative RT-PCR and Western blot were performed to verify the genetic pathways of NO release.ResultsAfter hours 24 of cell seeding, NO release was slowly enhanced by the increase of dot diameter from 10 nm up to 50 nm, mildly enhanced to a medium level at 100 nm, and increase rapidly to a high level at 200 nm. The temporal enhancement of NO release dropped dramatically on day 3. On day 5, a topology-dependent profile was established that maximized at 50 nm and dropped to control level at 200 nm. The NO releasing profile was closely associated with the expression patterns of genes associated with Endothelial nitric oxide synthase (eNOS) pathway [GPCR, PI3K, Akt, Bad, Bcl-2, NFκB(p65), eNOS], but less associated with Inducible nitric oxide synthase (iNOS) pathway (TNF-α, ILK, Akt, IκBα, NFκB, iNOS). Western blotting of Akt, eNOS, iNOS, and NFκB further validated that eNOS pathway was modulated by nanotopology.ConclusionsBased on the findings of the present study, 50, 100 nm can serve as the suitable nanotopography patterns for cardiac implant surface design. These two nanodot arrays promote NO secretion and can also promote the vascular smooth muscle relaxation. The results of this study can improve the heart stent design in the medical treatments.

Rap1 promotes endothelial mechanosensing complex formation, NO release and normal endothelial function.

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.

Immunomodulatory effects of clove (Syzygium aromaticum) constituents on macrophages: In vitro evaluations of aqueous and ethanolic components

The present work sought to investigate potential suppressive effects on mouse macrophages by in vitro treatment with clove (Syzygium aromaticum) ethanolic extracted essential oil (containing eugenol) or its water-soluble extract. Using doses (ranging from 0.001–1000 µg/ml) of each material freshly prepared in the laboratory, cell survival and production of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 by the treated cells (that in all cases also had received LPS stimulation) were measured. Results indicated that, except at doses ≥100 µg/ml, viability was unaffected in all groups. NO release by LPS-stimulated macrophages was generally significantly suppressed by either material; in contrast, low (i.e. 0.001–1 µg/ml) doses of either extract class appeared to enhance NO release by non-LPS (unstimulated)-treated macrophages. Among LPS-stimulated cells, TNFα release was also significantly affected by each extract; the ethanolic extract was suppressive at all doses tested, while the aqueous material was so up to 1 µg/ml and then became stimulatory. In contrast, nearly every dose of either extract appeared to stimulate IL-6 release from the LPS-treated cells. Effects on IL-12 production were overall inconsistent; in general, the ethanolic extract tended to be stimulatory of production by the LPS-treated cells. The data for the aqueous material showed no discernable pattern of effect. The results suggest that clove extracts do not have a distinct cytotoxic activity, but do impart potential anti- and pro-oxidant effects in cells, depending on their concentrations and on the activation state of the macrophages themselves at the time of exposure to the extracts. The impact of the extracts on macrophage cytokine release also displays a pattern of dose-relatedness.

Administration of Telmisartan Reduced Systolic Blood Pressure and Oxidative Stress Probably Through the Activation of PI3K/Akt/eNOS Pathway and NO Release in Spontaneously Hypertensive Rats

We investigated the effects of telmisartan, the blocker of angiotensin II receptor 1, on the regulation of systolic blood pressure (SBP) and oxidative stress through endothelial nitric oxide (NO) release in spontaneously hypertensive rats (SHRs). SHRs randomly received placebo, oral feeding of telmisartan (5 mg/kg or 10 mg/kg) every day and Wistar-Kyoto rats (WKYs) served as normotensive control. The SBP of rat was measured before and weekly thereafter. After a total of 8-week treatment, rats were killed for experimental measurements. Parameters that subject to measurements in isolated aorta endothelial cells include: NO concentration, protein expression levels of angiotensin II receptor 1, nitrotyrosine, 8-isoprostane, SOD, PI3K, Akt, AMPK and eNOS. In addition, L-NMMA, a general inhibitor of nitric oxide synthase, was also applied to test the inhibition of NO concentration. We found that SBPs were significantly lower in telmisartan therapy group than in placebo treated hypertensive rats and WKYs (p<0.05). The NO concentration was significantly higher in telmisartan-treated group with increased activity of the PI3K/Akt pathway and activated eNOS signaling. Blockade of Akt activity reversed such effects. Activation of AMPK also contributed to the phosphorylation of eNOS. L-NMMA treatment reduced less NO concentration in SHR rats than the telmisartan co-treated groups. Oxidative stress in SHRs was also attenuated by telmisartan administration, shown by reduced formation of nitrotyrosine, 8-isoprostane, and recovered SOD protein level. Telmisartan enhanced NO release by activating the PI3K/Akt system, AMPK phosphorylation and eNOS expression, which attenuated the blood pressure and oxidative stress in SHRs.

Effects of angiotensin receptor blockers on endothelial nitric oxide release: the role of eNOS variants

• Angiotensin II receptor blockers improve endothelial cell-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide synthase (eNOS) function. • The key finding from this study is that angiotensin II receptor blockers (ARBs) differentially enhanced nitric oxide (NO) release in a manner influenced by certain genetic variants of eNOS. This finding provides new insights into the effects of ARBs on endothelial cell-dependent vasodilation and eNOS function that are of high importance in vascular medicine and clinical pharmacology. AIM Angiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk. The effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 µm, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 µm) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism. RESULTS All of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n= 4-5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n= 4-5 in all eNOS variants) than increases observed with other ARBs. The ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.

Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells

The reagent 4,5-diaminofluorescein (DAF-2) is a widely utilized and sensitive fluorescent probe for real-time assessment of nitric oxide (NO) production. In this study we investigated the feasibility of using DAF-2 for detection of NO release from EA.hy 926 human endothelial cells stimulated with plant polyphenols. Flavonoids have recently gained much interest because of reported beneficial effects on vasodilatation, which have been ascribed to stimulation of endothelial NO production. DAF-2 shows moderate fluorescence, and because certain phenolic compounds quench fluorescence or fluoresce themselves, we utilized liquid chromatography to avoid interference. Our investigations with (+)-catechin and trans-resveratrol as test phenolic compounds revealed various previously undescribed principal methodologic pitfalls and limitations. Under assay conditions (+)-catechin displayed a highly significant increase in fluorescence intensity so that a control of test compound stability is advisable. Moreover, DAF-2 was subject to conversion to triazolofluorescein (DAF-2T) under certain assay and storage conditions; thus control of spontaneous reagent conversion is advisable. Finally, formation of DAF-2T was dose-dependently inhibited by polyphenols to a degree consistent with their free radical scavenging activity. The inhibition of DAF-2T generation seems to contradict previous reports on enhanced NO release from endothelial cells by (+)-catechin and resveratrol. Therefore, the planning of experiments involving NO measurement in biological systems and interpretation of results requires substantial scrutiny.