Stimulated Nitric Oxide Release Research Articles

NIR-Triggered Release of Nitric Oxide by Upconversion-Based Nanoplatforms to Enhance Osteogenic Differentiation of Mesenchymal Stem Cells for Osteoporosis Therapy.

Stem cell therapy is an attractive approach to bone tissue regeneration in osteoporosis (OP); however, poor cell engraftment and survival within injured tissues limits its success in clinical settings. Nitric oxide (NO) is an important signaling molecule involved in various physiological processes, with emerging evidence supporting its diverse roles in modulating stem cell behavior, including survival, migration, and osteogenic differentiation. To control and enhance osteogenic differentiation of mesenchymal stem cells (MSCs) for OP therapy, we designed a near-infrared (NIR) light-triggered NO-releasing nanoplatform based on upconversion nanoparticles (UCNPs) that converts 808-nm NIR light into visible light, stimulating NO release by light control. We demonstrate that the UCNP nanoplatforms can encapsulate a light-sensitive NO precursor, Roussin's black salt (RBS), through the implementation of a surface mesoporous silica coating. Upon exposure to 808-nm irradiation, NO is triggered by the controlled upconversion of UCNP visible light at the desired time and location. This controlled release mechanism facilitates photoregulated differentiation of MSCs toward osteogenic lineage and avoids thermal effects and phototoxicity on cells, thus offering potential therapeutic applications for treating OP invivo. Following the induction of osteogenic differentiation, the UCNP nanoplatforms exhibit the capability to serve as nanoprobes for the real-time detection of differentiation through enzymatic digestion and fluorescence recovery of UCNPs, enabling assessment of the therapeutic efficacy of OP treatment. Consequently, these UCNP-based nanoplatforms present a novel approach to control and enhance osteogenic differentiation of MSCs for OP therapy, simultaneously detecting osteogenic differentiation for evaluating treatment effectiveness.

Oral curcumin phytosome supplementation improves anthropometric measures of adiposity and enhances endothelial function in rats on a high-fat-diet regimen

Objectives: Curcumin has a protective role in endothelial function and nitric oxide (NO) production in animal models of different diseases; however, the role of curcumin on aortic reactivity in rats placed on a high-fat diet (HFD) remains unclear. This study aims to determine whether oral curcumin phytosome supplementation can reduce adiposity and enhance endothelial function. Materials and Methods: Rats were assigned to one of three groups: normal diet (ND), HFD for 20 weeks, and HFD supplemented with curcumin phytosome (HFD + Curcumin). Anthropometric measures were recorded weekly for the three groups, until the end of the feeding regimen. After 20 weeks of feeding on HFD, myographic investigations were conducted on thoracic aortic rings dissected from HFD and HFD + Curcumin rats. The response to high potassium chloride (KCl), incremental doses of phenylephrine (Phe) before and after L-NAME treatment, acetylcholine (ACh), or sodium nitroprusside (SNP), was evaluated. ACh-induced relaxation was also assessed in HFD + Curcumin rats, after preincubation with chromium III-mesoporhyrin. Results: HFD rats exhibited increased adiposity measures, some of which were negatively correlated with vasorelaxation response to ACh. HFD + Curcumin rats had reduced anthropometric measures, compared to HFD rats. Aortic rings from HFD and HFD + Curcumin rats exhibited comparable contractile responses to KCl and Phe. The difference in contractile response to Phe before and after L-NAME incubation was greater for HFD + Curcumin rats. ACh induced greater vasorelaxant responses in HFD + Curcumin rats. There was no group difference in the relaxant response to SNP. In HD + Curcumin rats, chromium III mesoporphyrin significantly reduced ACh-induced relaxations. Conclusion: Oral curcumin phytosome supplementation could reduce adiposity in rats placed on an HFD and may have enhanced basal and stimulated NO release from the endothelium, and heme oxygenase-1 may partly mediate this curcumin protective role. This study provides evidence that this curcumin formulation, taken as a daily supplement, may be effective in providing some protection against adiposity-associated adverse cardiovascular disorders.

Allyl Isothiocianate Induces Ca2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species Production in the Human Cerebrovascular Endothelial Cell Line hCMEC/D3.

Nitric oxide (NO) represents a crucial mediator to regulate cerebral blood flow (CBF) in the human brain both under basal conditions and in response to somatosensory stimulation. An increase in intracellular Ca2+ concentrations ([Ca2+]i) stimulates the endothelial NO synthase to produce NO in human cerebrovascular endothelial cells. Therefore, targeting the endothelial ion channel machinery could represent a promising strategy to rescue endothelial NO signalling in traumatic brain injury and neurodegenerative disorders. Allyl isothiocyanate (AITC), a major active constituent of cruciferous vegetables, was found to increase CBF in non-human preclinical models, but it is still unknown whether it stimulates NO release in human brain capillary endothelial cells. In the present investigation, we showed that AITC evoked a Ca2+-dependent NO release in the human cerebrovascular endothelial cell line, hCMEC/D3. The Ca2+ response to AITC was shaped by both intra- and extracellular Ca2+ sources, although it was insensitive to the pharmacological blockade of transient receptor potential ankyrin 1, which is regarded to be among the main molecular targets of AITC. In accord, AITC failed to induce transmembrane currents or to elicit membrane hyperpolarization, although NS309, a selective opener of the small- and intermediate-conductance Ca2+-activated K+ channels, induced a significant membrane hyperpolarization. The AITC-evoked Ca2+ signal was triggered by the production of cytosolic, but not mitochondrial, reactive oxygen species (ROS), and was supported by store-operated Ca2+ entry (SOCE). Conversely, the Ca2+ response to AITC did not require Ca2+ mobilization from the endoplasmic reticulum, lysosomes or mitochondria. However, pharmacological manipulation revealed that AITC-dependent ROS generation inhibited plasma membrane Ca2+-ATPase (PMCA) activity, thereby attenuating Ca2+ removal across the plasma membrane and resulting in a sustained increase in [Ca2+]i. In accord, the AITC-evoked NO release was driven by ROS generation and required ROS-dependent inhibition of PMCA activity. These data suggest that AITC could be exploited to restore NO signalling and restore CBF in brain disorders that feature neurovascular dysfunction.

Development of an Automated, High‐Throughput Assay to Detect Angiotensin AT2‐Receptor Agonistic Activity by Nitric Oxide Measurement In Vitro

BackgroundThe angiotensin AT2‐receptor (AT2R) is a key component within the protective arm of the renin‐angiotensin system inducing vasodilation by nitric oxide (NO) production. Currently, no high‐throughput assay is available to identify AT2R agonists in vitro, which could explain the low number of AT2R selective ligands in drug development programs.ObjectiveTo design and validate a high‐throughput method for detection of AT2R activation in vitro based on NO release.MethodsHuman aortic endothelial cells (HAEC) were seeded into 96‐well plates at a density of 5000 cells per well. 48 hours after seeding, cells were loaded for 30 minutes with the NO probe, DAF‐FM diacetate (5µM). Cells were stimulated for 15 minutes with the AT2R‐agonist C21 (1µM) or with classical NO‐releasing agents as positive controls: bradykinin (BK, 0.1µM), acetylcholine (ACh, 1µM) or Ang‐(1‐7) (MasR agonist, 0.1µM). Cells were fixed and fluorescence signals captured by fluorescence microscopy with automated, built‐in image analysis (ImageXpress Pico, Molecular Devices, USA). AT2R‐transfected (AT2R‐CHO) or non‐transfected (NT‐CHO) Chinese hamster ovary cells were cultured and loaded with DAF‐FM diacetate according to the same protocol as HAEC. Concentration‐response curves were obtained for C21, BK, Ang II and Ang‐(1‐5) (0.1nM to 10µM each) using the automated protocol for ImageXpress Pico described above on AT2R‐CHO. To evaluate whether effects of the compounds in AT2R‐CHO were AT2R specific, the response to these compounds at the highest concentration (10µM) was also tested in NT‐CHO.ResultsUsing primary HAEC we could show that the automated, high‐throughput workflow was able to detect NO release by AT2R agonists as efficiently as by classical NO‐releasing agents [C21: 16.16 ± 6.84%; Ang‐(1‐7): 25.45 ± 3.37%, BK: 19.30 ± 2.92%; ACh: 24.01 ± 4.24%; one‐way ANOVA followed by Dunnett’s multiple comparison test, p≤0.05 vs control, ≥3 independent experiments for all compounds). In AT2R‐CHO, we detected that both C21 and Ang II (AT2R agonists) increased NO release in a concentration‐dependent manner, while BK had no effect (AT2R‐CHO lacks BK receptors) at any tested concentration. Surprisingly Ang‐(1‐5), a degradation product of Ang II thought to be inactive, induced NO release in a concentration‐dependent manner in AT2R‐CHO. In NT‐CHO, none of the compounds stimulated NO release, indicating that the effects by C21, Ang II and Ang‐(1‐5) in AT2R‐CHO were AT2R specific.ConclusionMeasurement of NO release from AT2R‐CHO or HAEC by DAF‐FM diacetate in an automated way is suitable as high‐throughput assay for the identification of AT2R‐agonistic compounds in vitro. Application of the assay revealed that Ang‐(1‐5), which has been regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.

English

Many patients in Africa often utilize herbal medicine for the management of hyperactive gut disorders such as diarrhea and abdominal colic. There is therefore a need to scientifically evaluate efficacy of this herbal remedies. This study investigated the myorelaxant effect of Carissa edulis (Forssk.) Vahl and its possible mechanism of action using isolated rabbit jejunum preparations. Pieces of jejunum were isolated from adult New Zealand white rabbits. They were then mounted in an organ bath containing Tyrode’s solution. The rate and force of jejunal contraction were recorded and analyzed using a Powerlab that was coupled to Chart5 software for windows. The effects of the extract (0.1-10.0 mg/ml) on spontaneous contraction were investigated. Its effect (3.0 mg/ml) was also studied in the presence of LG-NAME (nitric oxide synthase inhibitor), methylene blue (soluble guanylyl cyclase inhibitor) and propranolol (non-selective β adrenergic receptors antagonist). The extract dose-dependent significantly decreased the force but not the rate of spontaneous rabbit jejunal contraction. Among the blockers used, only LG -NAME significantly blocked the effect of the extract. Aqueous root bark extracts of C. edulis possess significant myorelaxant effect on isolated rabbit jejunum. This appears to be mediated through stimulation of nitric oxide release by nitric oxide synthase. Key words: Carissa edulis, diarrhea, jejunum, motility, myorelaxant.

Hiroaki Shimokawa.

Hiroaki Shimokawa.

Wavelength-dependence of vasodilation and NO release from S-nitrosothiols and dinitrosyl iron complexes by far red/near infrared light

Far red/near infrared (R/NIR) energy is a novel therapy, but its mechanism of action is poorly characterized. Cytochrome c oxidase (Cco) of the mitochondrial electron transport chain is considered the primary photoacceptor for R/NIR to photolyze a putative heme nitrosyl in Cco to liberate free nitric oxide (NO). We previously observed R/NIR light directly liberates NO from nitrosylated hemoglobin and myoglobin, and recently suggested S-nitrosothiols (RSNO) and dinitrosyl iron complexes (DNIC) may be primary sources of R/NIR-mediated NO. Here we indicate R/NIR light exposure induces wavelength dependent dilation of murine facial artery, with longer wavelengths (740, and 830 nm) exhibiting reduced potency when compared to 670 nm. R/NIR also stimulated NO release from pure solutions of low molecular weight RSNO (GSNO and SNAP) and glutathione dinitrosyl iron complex (GSH-DNIC) in a power- and wavelength-dependent manner, with the greatest effect at 670 nm. NO release from SNAP using 670 was nearly ten-fold more than GSNO or GSH-DNIC, with no substantial difference in NO production at 740 nm and 830 nm. Thermal effects of irradiation on vasodilation or NO release from S-nitrosothiols and DNIC was minimal. Our results suggest 670 nm is the optimal wavelength for R/NIR treatment of certain vascular-related diseases.

Modulation of Nitric Oxide Release in Human Umbilical Vein Endothelial Cells by Myristolated‐PKC Epsilon Activator/Inhibitor Peptides

Ischemia/reperfusion (I/R) injury plays a critical role in endothelial dysfunction as a consequence of limited nitric oxide (NO) bioavailability and excess generation of reactive oxygen species (ROS) generated by endothelial nitric oxide synthase (eNOS) during reperfusion. Activated protein kinase C epsilon (PKCɛ) binds to a specific receptor for activated C kinase (RACK‐1), facilitating its translocation from the cytosol to the cell membrane for phosphorylation of targets, such as eNOS, at serine‐1177 to augment activity. Previous in vitro and in vivo animal studies have shown that PKCɛ activation stimulates eNOS activity, increasing NO release; whereas PKCɛ inhibition using a peptide inhibitor that interferes with its interaction with RACK‐1 results in decreased NO release. However, the modulation of PKCɛ‐mediated eNOS activity has yet to be demonstrated in human cells. In this study, we aim to demonstrate enhancement and attenuation of NO release in human umbilical vein endothelial cells (HUVECs) using cell‐permeable, myristic‐acid conjugated PKCɛ activator (Myr‐PKCɛ+), Myr‐HDAPIGYD (MW=1097 g/mol), and inhibitor (Myr‐PKCɛ−), Myr‐EAVSLKPT (MW=1054 g/mol), peptides respectively. Single‐donor HUVECs at passages 3–4 were grown to confluence in 6‐well (35 mm) plates. NO release was measured in real time using a calibrated NO electrode following the administration of 10 μM Myr‐PKCɛ+ or 10 μM Myr‐PKCɛ− treatments in the absence or presence of 10 μM acetylcholine (Ach) stimulation of NO release. Basal NO release (375±175 pM) was determined by measuring the difference between wells with and without cells (n=4). Myr‐PKCɛ+ increased NO levels by 36±13 pM (p<0.01, n=5) and Myr‐PKCɛ− decreased NO levels by 41±55 pM, p<0.01, n=5) significantly compared to baseline in the absence of Ach stimulation. In the presence of 10 μM Ach only, NO release significantly increased by 61±15 pM above baseline (p<0.01, n=5). In Ach‐stimulated cells, there was an additional significant increase in NO release by 70±23 pM (p<0.01, n=5) when treated with Myr‐PKCɛ+ compared to baseline, although there was no significance compared to the initial Ach‐stimulated NO release. Following initial 10 μM Ach administration, Myr‐PKCɛ− significantly reduced NO release by 48±16 pM, (p<0.01, n=5) compared to baseline. These results suggest Myr‐PKCɛ+ presumably increases NO release via activation of eNOS, whereas Myr‐PKCɛ− attenuates eNOS activity by inhibiting its phosphorylation in HUVECs; thus, eNOS modulation by these peptides demonstrate comparable effects across species. Elucidating the role of eNOS modulation is important for developing therapeutic interventions in the context of human organ I/R injury, in which uncoupled eNOS produces ROS instead of NO. Future studies will evaluate the effects of Myr‐PKCɛ+ and Myr‐PKCɛ− on NO release in HUVECs during reperfusion following prolonged ischemia compared to non‐ischemic controls.Support or Funding InformationThis study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Biomedical Sciences at Philadelphia College of Osteopathic Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of Mitochondrial KATP Channels by Diazoxide Stimulates Vascular Repair‐Relevant Functions of Human CD34+ Cells by Activating Endothelial Nitric Oxide Synthase

Diazoxide is a selective activator of mitochondrial ATP‐sensitive potassium channel (mitoKATP), which causes mitochondrial depolarization, and is known to be protective in ischemic conditions. Bone marrow derived CD34+ hematopoietic stem/progenitor cells have vascular‐reparative functions and are now being tested for their potential benefit for the treatment of ischemic vascular diseases in clinical trials. We hypothesized that diazoxide stimulates vascular reparative functions of CD34+ cells by mitochondrial mechanisms. The hypothesis was tested by determining the effects of diazoxide on the vascular repair‐relevant functions of CD34+ cells in vitro, and evaluated the mitochondrial mechanisms involved in the beneficial response. Peripheral blood was obtained from healthy individuals (n=58) and mononuclear cells (MNCs) were separated by Ficoll centrifugation. MNCs were enriched for lineage negative cells, which were further enriched for CD34+ cells by immunomagnetic selection. Proliferation and migration functions of CD34+ cells were evaluated by using BrdU‐ELISA, and by Chemotaxis assay, respectively. Mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS), and intracellular nitric oxide (NO) levels were evaluated by flow cytometry using JC‐1, mitoSOX and DAF‐FM, respectively. Diazoxide stimulated proliferation and migration of CD34+ cells in basal conditions, and potentiated these responses induced by hypoxia‐regulated factors, stromal‐derived factor‐1a (SDF) or vascular endothelial growth factor (VEGF), and in 5‐hydroxydecanoate (100 μM) (5HD)‐sensitive manner (P<0.05, n=6). Diazoxide (10 μM) induced mitochondrial depolarization of cells and decreased JC‐1 red fluorescence (P<0.05, n=8, vs untreated) that was reversed by 5HD. Diazoxide elevated intracellular NO levels that accompanied increased cGMP levels. NO release was blocked by a nonspecific NOS inhibitor, L‐NAME (300 μM) or by eNOS‐selective inhibitor, L‐N5‐(1‐Iminoethyl) ornithine (NIO, 10 μM) (P<0.01, n=6), but not by nNOS‐selective inhibitor, N‐ω‐Propyl‐L‐arginine (NPA, 50 nM). NO generation by diazoxide was sensitive to treatment with Ca2+ chelator BAPTA (10 μM), Akt inhibitor, triciribine (5 μM), or PI3K inhibitor, LY294002 (30 μM) (P<0.05, n=6). This was further confirmed by increased eNOS phosphorylation at Ser1177 or dephosphorylation at Thr495 (P<0.05, n=6). Diazoxide increased mitochondrial superoxide levels that was abolished by mitoTEMPOL (100 μM) (P<0.01, n=6), and interestingly, it was partially inhibited by L‐NAME or NPA (P<0.01, n=5) but not by NIO. Effects of diazoxide on proliferation and migration of CD34+ cells (P<0.05 vs untreated, n=6) were inhibited by L‐NAME (P<0.01, n=6) or by mitoTEMPOL (P<0.05, n=4). In conclusion, diazoxide stimulated vascular‐repair‐relevant functions of CD34+ cells in NO and ROS‐dependent mechanisms. Mitochondrial depolarization by diazoxide generates ROS, which in turn stimulates NO release by kinase‐ and Ca2+‐dependent activation of eNOS. Intriguingly, nNOS‐dependent generation of mitochondrial ROS contributes to the activation of eNOS.

Hydrogen sulfide, an enhancer of vascular nitric oxide signaling: mechanisms and implications.

Nitric oxide (NO) vascular signaling has long been considered an independent, self-sufficient pathway. However, recent data indicate that the novel gaseous mediator, hydrogen sulfide (H2S), serves as an essential enhancer of vascular NO signaling. The current article overviews the multiple levels at which this enhancement takes place. The first level of interaction relates to the formation of biologically active hybrid S/N species and the H2S-induced stimulation of NO release from its various stable "pools" (e.g., nitrite). The next interactions occur on the level of endothelial calcium mobilization and PI3K/Akt signaling, increasing the specific activity of endothelial NO synthase (eNOS). The next level of interaction occurs on eNOS itself; H2S directly interacts with the enzyme: sulfhydration of critical cysteines stabilizes it in its physiological, dimeric state, thereby optimizing eNOS-derived NO production and minimizing superoxide formation. Yet another level of interaction, further downstream, occurs at the level of soluble guanylate cyclase (sGC): H2S stabilizes sGC in its NO-responsive, physiological, reduced form. Further downstream, H2S inhibits the vascular cGMP phosphodiesterase (PDE5), thereby prolonging the biological half-life of cGMP. Finally, H2S-derived polysulfides directly activate cGMP-dependent protein kinase (PKG). Taken together, H2S emerges an essential endogenous enhancer of vascular NO signaling, contributing to vasorelaxation and angiogenesis. The functional importance of the H2S/NO cooperative interactions is highlighted by the fact that H2S loses many of its beneficial cardiovascular effects when eNOS is inactive.

Vascularization and VEGF expression in bovine yolk sacs: Impact of reproductive techniques

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.

Klotho modulates FGF23-mediated NO synthesis and oxidative stress in human coronary artery endothelial cells.

Chronic kidney disease (CKD) is a state of Klotho deficiency and excess of the phosphaturic hormone fibroblast growth factor 23 (FGF23). Both dysregulations were shown to be associated with endothelial dysfunction in humans, but direct vascular effects of FGF23 remain largely elusive. In vitro experiments were performed to assess the effects of FGF23 (10ng/mL) in relation to its co-receptor Klotho on nitric oxide (NO) synthesis and reactive oxygen species (ROS) formation and detoxification in human coronary artery endothelial cells (HCAEC). Membrane-bound Klotho is expressed in HCAEC, and FGF23 increases the expression of the Klotho shedding protease ADAM17, and consequently the secretion of soluble Klotho. FGF23 activates FGF receptor 1 and stimulates NO release via Akt-dependent activation of endothelial NO synthase (eNOS). Both FGF receptor (FGFR)-dependent ROS formation via activation of NADPH oxidase 2 (Nox2) as well as ROS degradation via superoxide dismutase 2 (SOD2) and catalase (CAT) is stimulated by FGF23. Pre-incubation with a Klotho inhibitor blunts the FGF23-stimulated Akt-eNOS activation and NO synthesis, and decreases ROS degradation by blocking SOD2 and CAT enzymes, whereas FGF23-stimulated ROS synthesis via Nox2 is unaffected, resulting in low NO bioavailability and increased oxidative stress. Our data indicate that in the presence of Klotho, FGF23 induces NO release in HCAEC and its stimulating effects on ROS production are counterbalanced by increased ROS degradation. In states of Klotho deficiency, e.g., CKD, FGF23-mediated NO synthesis is blunted and ROS formation overrules ROS degradation. Thus, FGF23 excess may primarily promote oxidative stress and thus endothelial dysfunction.

Nebivolol and Endothelial Dysfunction in Patients With Essential Hypertension: A Reputation Saver of β‐Blockers?

We have read with great interest the recently published article by Neuman and coworkers1 in which they hypothesized that nebivolol, a selective β1-antagonist that stimulates nitric oxide (NO), improves endothelial function in African Americans with hypertension when compared with metoprolol. In that well-designed study, the authors found evidence for NO bioavailability at rest during treatment with both nebivolol and metoprolol succinate in hypertensive African American patients, with a clear trend for a greater contribution of NO during nebivolol therapy. Moreover, after combined blockade of NO and endothelium-derived hyperpolarizing factor (EDHF), there was significantly greater vasoconstriction during nebivolol compared with metoprolol therapy, suggesting a greater contribution of both NO and EDHF combined to resting vasomotor tone during nebivolol treatment. Moreover, the contribution of NO to exercise-induced vasodilation was greater during treatment with nebivolol compared with metoprolol succinate. Nebivolol is a third-generation, β1-adrenergic receptor antagonist that has vasodilatory properties both in animals and humans independent of β1-receptor antagonism and related to β3-receptor agonist effects.2-5 It is a racemic mixture of D- and L-enantiomers, of which D-nebivolol is considered to be a highly selective β-adrenergic receptor antagonist.5-8 It was demonstrated that nebivolol stimulates NO release through both β3-receptor and adenosine triphosphate–dependent, P2Y-receptor activation.3, 9-12 In a well-designed clinical study, Tzemos and coworkers13 analyzed whether nebivolol could improve endothelial dysfunction in patients with essential hypertension. They showed that nebivolol/bendrofluazide increased both stimulated and basal endothelial NO release, whereas, for the same degree of blood pressure control, atenolol/bendrofluazide had no effect on NO bioactivity; therefore, they concluded that nebivolol may offer additional vascular protection in treating hypertension.13 Moreover, Oelze and coworkers14 demonstrated that nebivolol but not metoprolol improved endothelial function and reduced vascular oxidative stress in an experimental model of AngII-induced hypertension. These effects were associated with a normalization of the expression of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits Nox1, Nox2, p22phox, p47phox, p67phox, and Rac1 and an inhibition of NOS III uncoupling in a rat hypertension model. More importantly, they found that nebivolol but not metoprolol inhibited activation of vascular NADPH oxidase and were able to dissociate an already assembled and active membrane-associated NADPH oxidase complex. These observations indicate that nebivolol, in addition to its β1-receptor–blocking and NO-releasing effects, possesses substantial inhibitory effects on vascular oxidative stress, all of which may beneficially influence endothelial dysfunction because of oxidative stress in the setting of arterial hypertension.14 It is well-known that essential hypertension is associated with endothelial dysfunction, and with greater oxidative stress producing free oxygen radicals that contribute to the decrease in NO bioavailibility. Elevated plasma levels of malondialdehyde, a final product of lipid peroxidation and an index of oxidative stress, have been demonstrated in patients with essential hypertension.15, 16 Concordantly, our study group previously found that nebivolol, differently from metoprolol, improved oxidative stress and insulin sensitivity, decreased plasma sP-selectin, and increased adiponectin levels in hypertensive patients.17 From that standpoint, we concluded that nebivolol may significantly improve endothelial dysfunction in hypertensive patients. However, our study population had no African American patients, unlike the patient population from the study by Neumann and coworkers. In conclusion, nebivolol has beneficial effects on endothelial function mainly by increasing NO bioavailability. We strongly believe that although standard β-blockers are falling out of favor nowadays, perhaps the new generation of β-blockers such as nebivolol may “rescue” the reputation of these agents in the near future. There are no conflicts of interest to declare.

Dead Lactobacillus plantarum Stimulates and Skews Immune Responses toward T helper 1 and 17 Polarizations in RAW 264.7 Cells and Mouse Splenocytes.

This study was undertaken to evaluate the immunomodulatory effect of dead nano-sized Lactobacillus plantarum (nLp) in RAW 264.7 cells and murine primary splenocytes. nLp is a dead, shrunken, processed form of L. plantarum nF1 isolated from kimchi (a traditional Korean fermented cabbage) and is less than 1 μm in size. It was found that nLp treatment stimulated nitric oxide (NO) production more in RAW 264.7 macrophages than pure live L. plantarum (pLp), and that the stimulatory properties were probably largely derived from its cell wall. In addition, nLp induced murine splenocyte proliferation more so than pLp; in particular, a high dose of nLp (1.0 X 10(11) CFU/ml) stimulated proliferation as much as lipopolysaccharide at 2 μg/ml. Moreover, according to our cytokine profile results in splenocytes, nLp treatment promoted Th1 (TNF-α, IL-12 p70) responses rather than Th2 (IL-4, IL-5) responses and also increased Th17 (IL-6, IL-17A) responses. Thus, nLp stimulated NO release in RAW 264.7 cells and induced splenocyte proliferation more so than pLp and stimulated Th1 and Th17 cytokine production. These findings suggested that dead nLp has potential use as a functional food ingredient to improve the immune response, and especially as a means of inducing Th1/Th17 immune responses.

2-methoxyestradiol induces vasodilation by stimulating NO release via PPARγ/PI3K/Akt pathway.

The endogenous estradiol metabolite 2-methoxyestradiol (2-ME) reduces atherosclerotic lesion formation, while the underlying mechanisms remain obscure. In this work, we investigated the vasodilatory effect of 2-ME and the role of nitric oxide (NO) involved. In vivo studies using noninvasive tail-cuff methods showed that 2-ME decreased blood pressure in Sprague Dawley rats. Furthermore, in vitro studies showed that cumulative addition of 2-ME to the aorta caused a dose- and endothelium-dependent vasodilation. This effect was unaffected by the pretreatment with the pure estrogen receptor antagonist ICI 182,780, but was largely impaired by endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or by phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (WM). Moreover, 2-ME（10−7 ∼10−5 M）enhanced phosphorylation of Akt and eNOS and promoted NO release from cultured human umbilical endothelial cells (HUVECs). These effects were blocked by PI3K inhibitor WM, or by the transfection with Akt specific siRNA, indicating that endothelial Akt/eNOS/NO cascade plays a crucial role in 2-ME-induced vasodilation. The peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein expression were detected in HUVECs and the antagonist GW9662 or the transfection with specific PPARγ siRNA inhibited 2-ME-induced eNOS and Akt phosphorylation, leading to the impairment of NO production and vasodilation. In conclusion, 2-ME induces vasodilation by stimulating NO release. These actions may be mediated by PPARγ and the subsequent activation of Akt/eNOS cascade in vascular endothelial cells.

Endocannabinoid Stimulated Release of Nitric Oxide and its Mitochondrial Influence Triggering Vascular Pathology

Endocannabinoids, and their respective receptors, are involved in a host of cellular regulatory activities. In part, some of these mediated effects occur by way of stimulating constitutive nitric oxide release. This occurs in endothelia, certain white blood cells, microglia, and in similar invertebrate tissues, demonstrating that this is a conserved chemical messenger system. This endocannabinoid chemical messenger system, coupled to constitutive nitric oxide release, also appears to exert regulatory effects on mitochondrial energy associated processes, further substantiating its primordial history. In this regard, it appears to offer some beneficial actions in the occurrence of reperfusion injury and stroke. The mechanism envisioned is one initiated via a hypoxic event, which does not restore normalcy, then progresses to a pro-inflammatory state, and the resultant chronic condition manifests itself in a specific disorder. This fits nicely into a vascular-associated origin for Alzheimer’s Disease, whereby the proinflammatory state encompasses vessels that have endothelial gaps, providing for a compromised blood brain barrier, beta amyloid deposition, and enhanced white blood cell trafficking. In time, due to the physical progression of the events, Alzheimer’s Disease occurs.

The role of sex steroids on cellular events involved in vascular disease

In this work we checked the hypothesis whether estrone, progesterone, and testosterone are able to modulate the interactions between platelets, monocytes, and endothelial cells either under basal or inflammatory conditions. Using adhesion assays we demonstrated that pretreatment of endothelial cells with estrone, progesterone, or testosterone prevented monocytes and platelets adhesion induced by the proinflammatory agent bacterial lipopolysaccharide. The hormones reduced the expression of mRNA of ICAM-1, VCAM-1, and P-selectin, endothelial surface proteins that mediate monocytes and platelets adhesion respectively. Integrins are the main leukocyte proteins that allow firm adhesion. Using flow cytometry we showed that estrone treatment of monocytes reduced CD11b and CD11c expression, either under basal or injury (lipopolysaccharide) conditions. The three steroids inhibited platelet aggregation in a nitric oxide dependent manner. Platelet function was not affected by the steroid treatment. The molecular mechanisms of action exerted by the steroids included the participation of the intracellular signaling pathways PKC, MAPK, and PI3K, which selectively and differentially mediate the stimulation of nitric oxide release. We evidence that estrone, progesterone, and testosterone modulate monocyte and platelet adhesion to endothelial cells, events that play a major role in the initiation and progression of vascular lesions. The steroid action was evidenced under basal or inflammatory conditions. The mechanisms of action exerted by the steroids included stimulation of nitric oxide production and the participation of PKC, MAPK, and PI3K systems.

Panax ginseng Extract Rich in Ginsenoside Protopanaxatriol Attenuates Blood Pressure Elevation in Spontaneously Hypertensive Rats by Affecting the Akt-Dependent Phosphorylation of Endothelial Nitric Oxide Synthase

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a fundamental regulator of systemic blood pressure. Ginsenosides from Panax ginseng have been investigated in vitro for the molecular and biochemical mechanisms by which they stimulate NO release in vascular endothelial cells; however, little research has been done to confirm the physiological relevance of these in vitro studies. To address this research gap, the effects of a P. ginseng extract rich in ginsenosides from protopanaxatriol on spontaneously hypertensive rats (SHRs) was examined. Ginseng extract administration stimulated nongenomic Akt-mediated eNOS activation, enhanced NO production, improved vessel wall thickening, and alleviated hypertension in SHRs, confirming the physiological relevance of previous in vitro studies with ginsenosides.

Restoration of Endothelial Function via Enhanced Nitric Oxide Synthesis after Long-term Treatment of Raloxifene in Adult Hypertensive Rats

Raloxifene (CAS 84449-90-1, RAL), a selective estrogen receptor modulator (SERM) effective for the prevention of post-menopausal osteoporosis, also has been shown to acutely stimulate nitric oxide (NO) synthesis associated with improved endothelium-dependent relaxation. The effect of a 3-month RAL treatment (10 mg/kg/d) on basal blood pressure, measured via the carotid artery, and its challenge with increasing doses of intravenous bradykinin (1, 3 and 10 nmol/kg) was investigated. Furthermore, aortic NO bioavailability and relaxation in 9-month-old male and female ovarectomized (OVX) spontaneously hypertensive rats (SHR) was tested. Calcium ionophore stimulated NO release from aortic endothelial cells and aortic superoxide (O2-) production was directly assessed by using electrochemical nanosensors. Relaxation studies were performed with acetylcholine (10(-8) to 10(-5) mol/L) following precontraction with phenylephrine (10(-7) mmol/L). Whereas basal blood pressure (BP) was not significantly decreased in RAL treated SHR, the dose-dependent challenge with bradykinin induced an enhanced BP reduction in either gender. In contrast to female animals, aortic segments from RAL treated male animals showed significantly improved relaxaSHR) to 360 +/- 15 nmol/L. Vice versa, O2- was decreased from 110 +/- 15 to 22 +/- 1 nmol/L. In female SHR, ovarectomy led to an increase/decrease of NO/O2- from 130 +/- 5 to 180 +/- 10 nmol/L and 82 +/- 7 to 68 +/- 3 nmol/L, respectively. These effects were significantly amplified by RAL treatment (NO: 370 +/- 10 and O2-: 25 +/- 2 nmol/L). The results show that long-term treatment with RAL has beneficial affects on the cardiovascular system in old male and female OVX SHR via an increased NO bioavailability.

Nitric Oxide Supplementation in Postischemic Acute Renal Failure: Normotension Versus Hypertension

Nitric oxide (NO) has been suggested to play a pivotal role in ischemic acute renal failure (ARF) but there are controversies about its role in hypertensive and non hypertensive ischemic kidney. Multiple strategies including administration of exogenous NO donors have been shown to protect the kidney against toxic or ischemic injury, suggesting endothelial dysfunction as impaired NO generation due to ischemia. However, in postischemic kidney, NO derived from inducible nitric oxide synthase (iNOS) has been considered to enhance the tissue damage while iNOS inhibition decreased the tubular damage. It is well known decrease in basal production of NO in essential hypertension and that long lasting hypertension damages medium size and small-size blood vessels, therefore predisposes nephroangiosclerosis patients to ARF. Many studies have shown that long term stimulation of NO release in normotension improves renal haemodymnamics and kidney function in ischemic form of ARF. On the other hand, there are studies that have shown that NO synthesis stimulation has no effect or even worsens tubular damage in postischemic hypertensive kidney. Therefore, it seems likely that NO supplementation plays different role in postischemic renal damage development, beneficial in well preserved normotensive kidney and limited in postischemic hypertensive kidney due to disturbed tubuloglomerular response, vasoreactivity and kidney vascular structure.