Levels Of Nitric Oxide Release Research Articles

A Novel Stem Cell Model to Study Preeclampsia Endothelial Dysfunction.

Preeclampsia is a common pregnancy complication affecting 5% to 7% of all pregnancies worldwide annually. While the pathogenesis is not fully understood, maternal endothelium dysfunction is thought to be a central component to preeclampsia development. Studies to dissect maternal endothelial dysfunction, particularly on a patient-specific basis, are hampered by limited access to systemic primary endothelial cells (ECs). The objective of this study was to establish a replenishable, patient-specific in vitro EC model to allow robust mechanistic studies to dissect endothelial dysfunction in preeclampsia. Induced pluripotent stem cells (iPSCs) from three women with a history of normotensive pregnancies were differentiated into ECs. The established ECs were exposed to pooled sera from normotensive pregnancies, preeclamptic pregnancies, normotensive postpartum for non-pregnant comparison and controls. Endothelial functions including nitric oxide (NO) release, cell migration, tube formation and viability were evaluated. Levels of NO release were significantly lower after incubation with preeclamptic sera compared to the fetal bovine serum (FBS) control, and normotensive and non-pregnant (postpartum) sera treatments were also lower than FBS but higher than preeclamptic sera treatments. Tube formation and cell migration were also impaired with preeclamptic sera compared to FBS controls. Cell viabilities remained unaffected by any sera treatment. Consistent outcomes were obtained across all three patient-specific lines treated with the same pooled sera. Establishment of patient-derived iPSC-ECs treated with pregnancy sera serves as a novel model to explore the interplay between individual maternal endothelial health and circulating factors that lead to endothelial dysfunction in preeclampsia.

In-vitro cytotoxic and anti-inflammatory potential of Asparagus densiflorus meyeri and its phytochemical investigation.

Genus Asparagus is well known for its pharmacological activities and ethnopharmacological applications. In folk medicine, it is used in the management of several diseases such as diabetes, inflammations, and rheumatism. This work aimed to investigate the potential of Asparagus densiflorus meyeri root & aerial parts extracts as cytotoxic and anti-inflammatory and the investigation of their chemical profile. GC analysis & Folin-Ciocalteu and gravimetric methods were used respectively to estimate the lipoidal, phenolic, and saponin contents. MTT assay was conducted using two cell lines (MCF-7 & HepG2) to investigate the cytotoxic and anti-inflammatory activity using TNF-α stimulated MCF-7 cells through monitoring the level of nitric oxide release and NF-κB gene expression. Preliminary phytochemical investigation indicated that both extracted parts are equally rich in saponins, flavonoids, carbohydrates and/or glycosides, and sterols and/or triterpenes. Palmitic acid and β sitosterol represented the major saturated fatty acids and sterol, respectively. A significant cytotoxic activity against MCF-7 cells was recorded for DCM extract (IC50 26.13 μg/ml). All tested extracts showed a significant decrease in NO release and NF-κB gene expression thus it possesses a potential anti-inflammatory activity. A. densiflorus meyeri is considered a good candidate as a food supplement for protection from malignancy.

Antithrombotic and Antimicrobial Potential of S-Nitroso-1-Adamantanethiol-Impregnated Extracorporeal Circuit.

This study presents the utilization of a novel, highly lipophilic nitric oxide (NO) donor molecule, S-nitroso-1-adamantanethiol (SNAT), for developing an NO-emitting polymer surface aimed at preventing thrombus formation and bacterial infection in extracorporeal circuits (ECCs). S-nitroso-1-adamantanethiol, a tertiary nitrosothiol-bearing adamantane species, was synthesized, characterized, and used to impregnate polyvinyl chloride (PVC) tubing for subsequent in vivo evaluation. The impregnation process with SNAT preserved the original mechanical strength of the PVC. In vitro assessments revealed sustained NO release from the SNAT-impregnated PVC tubing (iSNAT), surpassing or matching endothelial NO release levels for up to 42 days. The initial NO release remained stable even after 1 year of storage at -20°C. The compatibility of iSNAT with various sterilization techniques (OPA Plus, hydrogen peroxide, EtO) was tested. Acute in vivo experiments in a rabbit model demonstrated significantly reduced thrombus formation in iSNAT ECCs compared with controls, indicating the feasibility of iSNAT to mitigate coagulation system activation and potentially eliminate the need for systemic anticoagulation. Moreover, iSNAT showed substantial inhibition of microbial biofilm formation, highlighting its dual functionality. These findings underscore the promising utility of iSNAT for long-term ECC applications, offering a multifaceted approach to enhancing biocompatibility and minimizing complications.

Glucosamine Hydrochloride and Glucosamine‐Gallic Acid Nanoparticles for the Treatment of Osteoarthritis: Synthesis, Antioxidant, and Anti‐Inflammatory

This study aimed to evaluate the antioxidant and anti‐inflammatory effects of glucosamine nanoparticles (GNPs) grafted with gallic acid (GNPs‐g‐GA). Glucosamine hydrochloride (G‐HCl) was produced from shrimp shell, and then GNPs synthesized using ionic gelation method. GNPs‐g‐GA was prepared by coupling GNPs with GA via 1‐ethy‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC) in combination with N‐hydroxysuccinimide (NHS) cross‐linking agents. The results indicated that the grafting of GA onto GNPs at different ratios increased the average size of the nanoparticles from 195.7 to 294.2 nm with various grafting degrees ranging from 73.3 to 146.4 mg GA/g GNPs‐g‐GA. The SEM images revealed the formation of spherical‐shaped GNPs‐g‐GA nanoparticles with approximate sizes ranging from 275.3 to 303.6 nm. The appearance of characteristic signals in the FT‐IR (C=C, C–O/C–C, and NH2) and 1H‐NMR (H‐2 and H‐6 at 6.95 ppm) spectra and the red shift in UV‐Vis spectrum provided further support of GNPs‐g‐GA successful synthesis. DPPH radical scavenging (from 20.0 to 70.4%) and ABTS radical scavenging (from 18.7 to 79.0%) activities and reducing power (nearly fivefold) sharply improved in GNPs‐g‐GA. Moreover, GNPs‐g‐GA was found nontoxic and drastically reduced the level of nitric oxide release and downregulated the synthesis of TNF‐α, IL‐1β, and IL‐6 in LPS‐induced RAW2647 murine macrophage cells through NF‐κB and MAPKs signaling pathways. Overall, these results suggested that the grafting of GNPs and GA is an effective strategy for the suppression of inflammation response and oxidation reaction in osteoarthritis.

Synthesis and antitumor activity evaluation of coumarin Mannich base derivatives.

Twenty-one new coumarin Mannich base derivatives (11a-u) were synthesized, which exhibited antiproliferation activities in HepG2 (liver cancer), A549 (lung cancer), MCF-7 (breast cancer), and HT-29 (colon cancer). Most of the target compounds showed the most potent activity against HepG2 cells compared with other cancer cells, compound 11g showed the strongest antiproliferative activity (2.10 μM) against HepG2, even superior to the positive control drug 5-FU(5.49 μM). The nitric oxide (NO) release of all compounds in HepG2 cells was determined, of which compound 11g showed high levels of NO release (10.8 μM). Notably, the solubility of compound 11g increased 13-fold compared with the lead 8. The preliminary cytotoxicity studies suggest that 11g had little effect on LO2 cells(normal liver cells, >50 μM). The effect of compound 11g on the apoptosis of HepG2 cells was also studied, and the results showed that the induction effect of compound 11g on apoptosis is a concentration-dependent manner. Our results indicate that compound 11g might be a promising lead for further studies.

Potent, Broad-Spectrum Antimicrobial Effects of S-Nitroso-N-acetylpenicillamine-Impregnated Nitric Oxide-Releasing Latex Urinary Catheters.

Although numerous prevention and intervention techniques have been developed to counteract catheter-associated urinary tract infections (CAUTIs), urinary catheters remain one of the most common sources of hospital-acquired infections. Nitric oxide (NO), a gaseous free radical responsible for regulating many physiological functions in the body, has gained immense popularity due to its potent, broad-spectrum antimicrobial activity, which is capable of combating medical device-associated infections. In this work, a straightforward solvent-swelling method was used to load the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) into commercial latex catheters (SNAP-UCs) for the first time. The effects of swelling catheters with different concentrations of SNAP solutions (25-125 mg/mL SNAP in tetrahydrofuran (THF)) were studied by measuring the NO release kinetics, SNAP loading, and SNAP leaching. SNAP-UCs impregnated with a 50 mg/mL SNAP-THF solution were found to maximize the amount of SNAP loaded into the latex (0.115 ± 0.009 mg SNAP/mg catheter) and showed physiological levels of NO release (>2 × 10-10 mol min-1 cm-2) over 7 days and minimal SNAP leaching (<2%). SNAP-UCs showed impressive in vitro contact-based and diffusible antimicrobial efficacy against three CAUTI-associated pathogens, reducing the viability of adhered and planktonic Escherichia coli, Proteus mirabilis, and Staphylococcus aureus by ∼98.0 to 99.1% (adhered) and 86.3-96.3% (planktonic) compared to control latex catheters. In vitro cytotoxicity against 3T3 mouse fibroblasts using a CCK-8 assay showed that SNAP-UCs were noncytotoxic (>90% viability). In summary, SNAP-UCs show stable, noncytotoxic NO release characteristics capable of potent, broad-spectrum antimicrobial activity, demonstrating great potential for reducing the devastating effects associated with CAUTIs.

Controlled light-induced gas phase nitric oxide release from S-nitrosothiol-doped silicone rubber films

The light induced nitric oxide (NO) release properties of S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) NO donors doped within polydimethylsiloxane (PDMS) films (PDMS-SNAP and PDMS-GSNO respectively) for potential inhaled NO (iNO) applications is examined. To achieve photolytic release of gas phase NO from the PDMS-SNAP and PDMS-GSNO films, narrow-band LED light sources are employed and the NO concentration in a N2 sweep gas above the film is monitored with an electrochemical NO sensor. The NO release kinetics using LED sources with different nominal wavelengths and optical power densities are reported. The effect of the NO donor loading within the PDMS films is also examined. The NO release levels can be controlled by the LED triggered release from the NO donor-doped silicone rubber films in order to generate therapeutic levels in a sweep gas for suitable durations potentially useful for iNO therapy. Hence this work may lay the groundwork for future development of a highly portable iNO system for treatment of patients with pulmonary hypertension, hypoxemia, and cystic fibrosis.

Alterations in HO-1 expression, heme oxygenase activity and endogenous NO homeostasis modulate antioxidant responses of Brassica nigra against nano silver toxicity

Silver nanoparticles (AgNPs) are one of the most widely-used nanomaterials, which are toxic and can cause physiological disorders in plants. The aim of the present study was to investigate whether a possible signaling link between heme oxygenase (HO) and nitric oxide (NO) is implicated in alleviating the toxicity of AgNPs as well as AgNO3. The results showed that exposure to 400 mg L−1 of AgNPs or AgNO3 reduced the chlorophyll content and the growth parameters in Brassica nigra. Data on Ag accumulation as well as the evaluation of lipid peroxidation and the H2O2 content in roots and shoots revealed that AgNP exerted more toxicity than AgNO3. Applying AgNP and AgNO3, respectively, increased HO transcripts by 87.5 and 37.3% and elevated the endogenous NO content 51.8 and 28.5%. The application of both hematin (as an inducer of HO) and sodium nitroprusside (SNP, as a NO donor) reversed the chlorosis and improved plant growth under AgNP and Ag+ ions stresses. Hematin decreased Ag accumulation in plants, indicating that this compound triggered an avoidance mechanism. Hematin and SNP enhanced the activities of antioxidant enzymes and proline accumulation, in parallel to increasing HO transcripts and NO release levels in the roots. ZnPPIX, as the inhibitor of HO, and cPTIO, as the specific NO scavenger, differentially blocked these effects. These findings revealed for the first time that HO might confer an increased tolerance to AgNP by activating the antioxidant systems, which was partially mediated by NO signal.

Anticancer efficacy of a nitric oxide-modified derivative of bifendate against multidrug-resistant cancer cells.

The development of multidrug resistance (MDR) not only actively transports a wide range of cytotoxic drugs across drug transporters but is also a complex interaction between a number of important cellular signalling pathways. Nitric oxide donors appear to be a new class of anticancer therapeutics for satisfying all the above conditions. Previously, we reported furoxan‐based nitric oxide‐releasing compounds that exhibited selective antitumour activity in vitro and in vivo. Herein, we demonstrate that bifendate (DDB)‐nitric oxide, a synthetic furoxan‐based nitric oxide‐releasing derivative of bifendate, effectively inhibits the both sensitive and MDR tumour cell viability at a comparatively low concentration. Interestingly, the potency of DDB‐nitric oxide is the independent of inhibition of the functions and expressions of three major ABC transporters. The mechanism of DDB‐nitric oxide appears to be in two modes of actions by inducing mitochondrial tyrosine nitration and apoptosis, as well as by down‐regulating HIF‐1α expression and protein kinase B (AKT), extracellular signal‐regulated kinases (ERK), nuclear factor κB (NF‐κB) activation in MDR cells. Moreover, the addition of a typical nitric oxide scavenger significantly attenuated all the effects of DDB‐nitric oxide, indicating that the cytotoxicity of DDB‐nitric oxide is as a result of higher levels of nitric oxide release in MDR cancer cells. Given that acquired MDR to nitric oxide donors is reportedly difficult to achieve and genetically unstable, compound like DDB‐nitric oxide may be a new type of therapeutic agent for the treatment of MDR tumours.

Improved hemocompatibility and analytical performance of intravascular oxygen sensors via nitric oxide (NO) releasing catheters

Event Abstract Back to Event Improved hemocompatibility and analytical performance of intravascular oxygen sensors via nitric oxide (NO) releasing catheters Elizabeth Brisbois1, Hang Ren2, Madeline Mccabe2, Terry C. Major1, Mark E. Meyerhoff2 and Robert H. Bartlett1 1 University of Michigan, Surgery, United States 2 University of Michigan, Chemistry, United States Introduction: Continuous monitoring of blood analytes (e.g., blood gases, pH, glucose, lactate, etc.) could provide valuable information needed for the diagnosis and treatment of patients. However, implantable biosensors and other blood-contacting devices suffer from two major clinical problems: clotting and infection. Thrombus formation on implantable sensors can severely impact their accuracy due to the metabolic activity of adhered cells altering the local levels of the analytes. One approach to improving hemocompatibility is the development of polymers that mimic the NO release from the normal endothelium (NO surface flux of 0.5-4x10-10 mol cm-2 min-1) that is a known potent inhibitor of platelet adhesion/activation and endogenous antimicrobial agent. One approach has been the development of NO releasing polymers by incorporation of NO donor molecules, such as S-nitrosothiols (RSNOs) [1]-[3]. However, many RSNO-based polymers have been limited by rapid leaching, low synthetic yields, and instability during storage. Recent work has shown that incorporating S-nitroso-N-acetylpenicillamine (SNAP) within hydrophobic polymers creates materials with long-term NO release and excellent storage stability [4]. In this study, a novel solvent swelling technique is used to load the walls of catheters with SNAP. The SNAP-based catheters are used to fabricate intravascular oxygen (PO2) sensors and their biocompatibility is evaluated in a rabbit model. Methods: The SNAP-based catheters were prepared by swelling silicone rubber catheters in a tetrahydrofuran solution containing SNAP. Control and SNAP-based catheters were used to fabricate the Clark-style amperometric PO2 sensors. The catheter lumen was filled with 0.15 M KCl in 0.1 M bicarbonate/carbonate buffer (pH 10). A Pt and a Ag/AgCl wire were inserted in the catheter lumen, and both ends of the catheter were sealed with silicone rubber. The PO2 sensors were soaked in 10 mM PBS, 100µM EDTA at 37 °C. NO release from the SNAP-based PO2 sensors under physiological conditions was determined via a chemiluminescence NO analyzer (Sievers280i, Boulder, CO). Sensors were calibrated by immersing in PBS and bubbling with O2 (0-100%). A potential of -0.7 V was applied to the Pt working electrode and the current was recorded. A rabbit model (sensors placed in veins) was used to evaluate the effects on thrombus formation and analytical PO2 sensing performance, as compared to a blood gas analyzer. After 7 h, the sensors were explanted, pictures were taken of the thrombus, and the 2D representation of the thrombus area was determined with the NIH ImageJ software. Results: The SNAP-based PO2 sensors can release NO under physiological conditions for up to 2 weeks (Fig. 1). During in vitro calibrations, the SNAP-based and control sensors had similar responses to various levels of oxygen in terms of sensitivity and response times (Fig. 2). Control (without NO release) and SNAP-based PO2 sensors were evaluated in a rabbit model. The SNAP-based PO2 sensors were found to accurately measure the PO2 levels in blood (within 10% of a standard benchtop blood gas analyzer). At the end of 7 h, the SNAP-based PO2 sensors were also found to have significantly reduced thrombus formation (as compared to controls). Conclusions: NO releasing catheters can be prepared with a novel solvent swelling technique that loads the walls of existing/commercial silicone rubber catheter tubing with SNAP. These SNAP-based catheters can be used to fabricate intravascular PO2 sensors with enhanced hemocompatibility properties and more accurate PO2 measurements. The NO release levels observed from the SNAP-based PO2 sensors are expected to also possess antimicrobial properties against bloodborne pathogens (e.g., S. aureus, S. epidermidis) [5],[6]. The SNAP solvent swelling technique also has the potential of improving the hemocompatibility of other blood-contacting devices (e.g., catheters, extracorporeal circuits, etc.). This work was supported in part by the University of Michigan Postdoctoral Translational Scholars Program (UL1TR000433) and NIH grant R01HL128337.

Synthesis and properties of polyvinylpyrrolidone films containing iron nitrosyl complexes as nitric oxide (NO) donors with antitumor and antiseptic activities

New homogeneous water-soluble polyvinylpyrrolidone composites containing iron sulfur nitrosyl complexes as nitric oxide donors with antitumor and antiseptic activities were synthesized. The morphology of the composites in the solid state was examined by X-ray diffraction and scanning electron microscopy. The study by small-angle X-ray scattering showed that samples of the composites do not contain 10–100-nm aggregates and that iron sulfur nitrosyl complexes are uniformly distributed in polymer matrices. The percentage of NO-releasing iron sulfur nitrosyl complexes in the composites was determined by ESR spectroscopy. An amperometric analysis demonstrated that the polymer composites decompose to yield NO within a few seconds after the dissolution in water without additional thermal and photoactivation. The dependences of the amount of NO and the maximum level of NO release on the duration of the storage of aqueous solutions for all of the synthesized films were determined at pH 7.0. Some of the synthesized polymer composites retain NO-donor activity of the starting pharmacologically active iron nitrosyl complexes and can be recommended to be used as targeted delivery systems of this class of compounds to biological targets.

Cell viability, adhesion and function of RAW 264.7 macrophages on fluorinated xerogel-derived nitric oxide permeable membrane for the application of cellular sensing.

Organically modified xerogels have an advantage over gas sensing applications due to their open, rigid structure and hydrophobicity. Here we evaluated the biocompatibility of xerogel-derived nitric oxide (NO) permeable membranes modified with fluorinated functional groups for application in cellular sensing by growing RAW 264.7 macrophages on them. We examined the cell viability, adhesion and growth of RAW 264.7 macrophages on NO permselective membrane and other cell-adhesive matrices, poly L-lysine and collagen. The surface roughness of each membrane was obtained from topographic atomic force microscopy (AFM) images. In addition, we measured the level of NO release of RAW 264.7 macrophages by lipopolysaccharide (LPS) stimulation using a Griess assay to confirm the function of cells. The fluorinated xerogel-derived membrane had a very smooth surface with rms roughness 2.1 Å and did not show cytotoxic effects in RAW 264.7 macrophages. As a result, the morphology and function of adhering RAW 264.7 macrophage showed no differences from those of other cell-adhesive membranes. Finally, we successfully detected NO release in RAW 264.7 macrophages stimulated by LPS, using a planar-type xerogel-derived NO sensor. Therefore, we suggest that fluorinated xerogel-derived membrane could be used as both a NO permeable and cell-adhesive membrane for cellular sensing applications.

Real-time evaluation of nitric oxide (NO) levels in cortical and hippocampal areas with a nanopore-based electrochemical NO sensor

Nitric oxide (NO) is an important biomolecule for regulating various brain functions, such as the control of neurovascular tone. NO, however, cannot be stored inside cells where NO is produced and immediately diffuses through the cellular membrane and decays rapidly, which makes the detection of NO extremely hard in an in vivo setting. We constructed an amperometric NO nanosensor and utilized it to directly measure NO release in the living brain. The NO nanosensor uses nanopores (pores with an opening radii <500nm) in which NO is oxidized at the porous platinum surface. The nanopore-based sensor was inserted vertically into the brains of anesthetized mice up to the end of the hippocampal CA 3 region, or to a depth of about 3mm. The sensor was slowly advanced in the brain in 0.5μm increments and in 0.05s temporal steps. Different levels of NO release were monitored by the nanopore NO sensor during the course of the penetration. The hippocampal CA3 region had the highest level of NO release, which was followed by CA2 and CA1 of the hippocampus and the cortex. The levels of NO release were not uniformly distributed within the cortical and hippocampal areas of living brain. In sum, the nanopore-based NO sensor was able to grossly measure NO contents within living brain in real time and with high sensitivity. This study may provide good insights about the relationship between the distributions of NOS-immunoreactive neurons and the directly measured levels of NO release in brain.

Impairment of both nitric oxide‐mediated and EDHF‐type relaxation in small mesenteric arteries from rats with streptozotocin‐induced diabetes

To investigate whether diabetes affects either or both nitric oxide (NO)-mediated and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in endothelium-dependent relaxation of mesenteric arteries from streptozotocin-induced diabetic rats. Wire myography was employed to examine endothelial function of mesenteric arteries. Superoxide levels were measured by L-012 and lucigenin-enhanced chemiluminescence. Western blotting was used to quantify protein expression levels. Superoxide levels were significantly increased in diabetic mesenteric arteries compared with normal arteries. Diabetes significantly reduced the sensitivity to the endothelium-dependent relaxant, acetylcholine (ACh) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA) + a soluble guanylate cyclase inhibitor (ODQ), the sensitivity to ACh was significantly decreased in the diabetic arteries compared with normal arteries, indicating an impaired EDHF-type relaxation despite increased expression of intermediate- and small-conductance calcium-activated potassium channels. Conversely, when the contribution of EDHF was inhibited with TRAM-34 + apamin + iberiotoxin, maximum relaxations to ACh were significantly decreased in diabetic compared with normal arteries, suggesting that the contribution of NO was also impaired by diabetes. Basal levels of NO release, indicated by contraction to L-NNA, were also significantly decreased in diabetic arteries. Western blot analysis demonstrated that diabetic arteries had an increased expression of Nox2, decreased pSer⁴⁷³ Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling. The contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer⁴⁷³ Akt and/or eNOS uncoupling.

Ramaline inhibits LPS‐induced NO release in macrophages by regulating MAPK and NF‐κB activities.

Lipopolysaccharide (LPS) induces innate immune responses through TLR4 signaling and activated macrophages produce various inflammatory mediators including Nitric oxide (NO). NO produced by inducible NO synthase (iNOS) mediates a number of pathophysiological processes. Therefore, inhibiting NO production in macrophages is a potential therapeutic target in inflammatory diseases. Herein, we demonstrate that ramalin, a newly discovered antioxidant that is extracted from polar lichen, inhibits production of inflammatory mediators in macrophage‐like Raw 264.7 cell line. RT‐PCR and Western blot analysis demonstrated that ramalin suppressed the level of NO release through down‐regulating both mRNA and protein levels of iNOS. Western blot analysis also revealed that ramalin abrogated LPS‐induced phosphorylation of ERK, p38, and JNK as well as the expression of p65. Attenuated activity of NF‐κB by ramalin was also confirmed by Luciferase promoter assay. In addition, not only the surface level of LPS‐induced TLR4 but also its mRNA and protein levels were decreased by ramalin. Together, these results suggest that ramalin reduces NO release in macrophage through down‐regulation of TLR4 signaling which in turn suppresses MAPK and NF‐κB activities, and it would be a possible therapeutic approach to the treatment of inflammatory diseases.

Membrane location of nebivolol contributes to antioxidant activity and endothelial nitric oxide release in Stroke-prone Hypertensive Rats

We tested the ability of nebivolol, a β1-selective blocker with direct vasodilating properties, to reverse endothelial dysfunction in Stroke-prone Hypertensive Rat (SHR). The effects of nebivolol on endothelial-dependent nitric oxide (NO) release were correlated with cellular redox state, NO synthase efficiency and molecular membrane localization. The effect of nebivolol on NO bioavailability, a key indicator of endothelial function, was measured using a porphyrinic nanosensor placed into the lumens of vascular rings from small peripheral arteries. As compared to normal rats, a marked deficiency (>40%, p < 0.01) in NO release was measured in SHR following stimulation with either acetylcholine or calcium ionophore. Nebivolol treatment restored the level of NO release in SHRs to that seen in control animals. In addition, greater NO release was seen with the enantiomer (l-nebivolol) that lacks β1-adrenergic activity (340 nM NO versus 180 nM NO for the β1-selective d-nebivolol). The mechanism for nebivolol activity may be due to its distinct membrane location that facilitates potent antioxidant scavenging properties, as determined by x-ray diffraction. The antioxidant activity of nebivolol and its enantiomers were not reproduced by other β-blockers at low concentrations. Nebivolol is a potent antioxidant that reversed eNOS uncoupling and restored endothelial-dependent NO release in arteries from hypertensive animals prone to stroke, independently of β1-blockade activity.

Sol–gel derived nitric oxide-releasing oxygen sensors

An amperometric sol-gel derived sensor that both releases nitric oxide (NO) and measures physiologically relevant concentrations of oxygen (PO2) is described. The sensor consists of a platinum electrode coated with an aminosilane/ethyltrimethoxysilane hybrid xerogel film. Hydrophilic polyurethane (HPU) is doped into the hybrid film to reduce sensor hydration time and increase oxygen permeability. Diazeniumdiolate NO donors are formed within the polymer matrix by exposing the cured film to high pressures of NO. These coatings release up to 7.2 pmol s(-1) cm(-2) of NO over the first 12 h and maintain detectable levels of NO release through 48 h. Sensors modified with HPU-doped, NO-releasing xerogels exhibit a linear response to O2 within 30 min of polarization at -0.65 V vs. Ag/AgCl, and have a sensitivity of approximately 6 nA/mmHg O2. The xerogel coating is stable in buffer solution with minimal fragmentation over 48 h. In vitro biocompatibility studies indicate that these materials effectively reduce platelet adhesion.

Impact of cirrhosis on the development of experimental hepatic metastases by B16F1 melanoma cells in C57BL/6 mice.

Metastases rarely occur in human livers with cirrhosis in clinical studies. We postulated that this phenomenon would also occur in experimental cirrhosis. Cirrhosis was established in C57BL/6 mice by carbon tetrachloride (CCl(4)) gastrogavage. B16F1 melanoma cells were injected into the mesenteric vein to induce hepatic metastases. Contrary to our postulate, there was greater than 4-fold increase in metastasis in animals with cirrhosis compared to controls. Intravital videomicroscopy showed that the hepatic sinusoids were narrower and more tumor cells were retained in the terminal portal vein (TPV) in cirrhotic livers. Immunohistochemistry demonstrated that the expression of vascular adhesion molecules was significantly increased in cirrhosis. Using confocal microscopy and the fluorescent nitric oxide (NO) probe 4,5-diaminofluorescein diacetate, a significantly lower level of NO release was detected in livers with cirrhosis both in basal conditions and after tumor cell arrest. Eight hours after mesenteric vein tumor cell injection, the percentage of apoptotic tumor cells in the sinusoids was 17% +/- 2% in livers with cirrhosis and 30% +/- 5% in normal livers. More mitotic and Ki-67 labeled tumor cells were seen in livers with cirrhosis. In conclusion, the changes in architecture and adhesion molecule expression in livers with cirrhosis may cause more tumor cells to arrest in the TPV. Lower levels of NO production may reduce apoptosis of B16F1 cells in livers with cirrhosis. As a result, these changes may promote the growth of metastasis in this cirrhotic model.

Estrogen induces nitric oxide production via activation of constitutive nitric oxide synthases in human neuroblastoma cells.

Although it is becoming increasingly evident that nitric oxide (NO) mediates some of estrogen's actions in the brain, the effects of estrogen on NO production through NO synthases (NOS) in neuronal cells have not yet been identified. Here we assessed changes in NO production induced by 17beta-estradiol (E2) in cells of neuronal origin using human SK-N-SH neuroblastoma cells, which we show express all three isoforms of NOS. Involvement of NOS isoforms in E2-induced NO production was examined using isoform-specific NOS inhibitors. E2 (10(-10)-10(-6) m) induced rapid increases in NO release and changes in endothelial NOS (eNOS) expression, which were blocked by ICI 182,780, an antagonist of estrogen receptors. Increased levels of NO release and NOS activity induced by E2 were blocked by N5-(1-Imino-3-butenyl)-L-ornithine, a neuronal NOS inhibitor, and N(5)-(1-Iminoethyl)-L-ornithine, an eNOS inhibitor, but not by 1400W, an inducible NOS inhibitor. These results demonstrate that E2-stimulated NO production occurs via estrogen receptor-mediated activation of the constitutive NOSs, neuronal NOS and eNOS. The E2-induced NO increase was abolished when extracellular Ca2+ was removed from the medium or after the addition of nifedipine, an L-type channel blocker, and was partially inhibited using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester itself also caused an increase in NO release that was blocked by 1400W, suggesting that inducible NOS mediates this response. Together these data reveal that constitutive NOS activities are responsible for E2-induced NO production in neuroblastoma cells and that differential activation of NOS isoforms in these cells occurs in response to different treatments.

Food restriction attenuates age-related increase in the sensitivity of endothelial cells to oxidized lipids.

Age-related endothelial dysfunction has been suggested to play a role in atherogenesis. Food restriction (FR) has been shown to retard the development of atherosclerosis. The goal of this report is to assess the effect of aging and FR on endothelial functions, including the release of endothelial nitric oxide (NO) and the adhesion of mononuclear cells (MNCs) to endothelial cells (ECs). ECs were obtained from the aorta of young mice fed ad libitum (Y-AL), old mice fed ad libitum (O-AL), or a food-restricted diet (O-FR). When compared with those obtained from Y-AL and O-FR mice, ECs obtained from O-AL mice decreased the basal level of NO release and increased the basal level of peroxynitrite, superoxide, and hydrogen peroxide. In addition, ECs obtained from O-AL elevated the response to CuSO4-oxidized low-density lipoprotein (oxLDL). For example, incubation with oxLDL reduced NO release approximately 52% in ECs obtained from O-AL mice. In contrast, the same dose of oxLDL reduced NO release only approximately 40% in ECs obtained from Y-AL and O-FR mice. Moreover, the level of oxLDL-induced adhesion of MNCs and oxLDL-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 was markedly higher in ECs obtained from O-AL mice as compared with those obtained from Y-AL and O-FR mice. These results suggest that aging increases the sensitivity of ECs in response to oxLDL-reduced endothelial NO release and oxLDL-increased adhesion of MNCs to ECs. FR attenuates age-related increase in the sensitivity of ECs to oxLDL, which might be responsible, at least in part, for the antiatherogenic action of FR.