Oxidases and oxygenases in regulation of vascular nitric oxide signaling and inflammatory responses

Nitric oxide (^aNO) is a freely diffusible inter-and in tracellular messenger produced by a variety of mammalian cells including vascular endothelium, neurons, smooth muscle cells, macrophages, neutrophils, platelets, and pulmonary epithelium. In smooth muscle cells, platelets, and neutrophils, ^aNO raises intracellular cyclic guanasine 5′-monophosphate levels by reacting with the actalytic heme domain of guanylate cylase, to activate it, thus leading to vasorelaxation, inhibition of platelet aggregation and inhibition of platelet and inflammatory cell adhesion to endothelium. The physiologic actions of ^aNO are highly dependent on changes in steady-state concentrations of reactive species and tissue-oxidant defense mechanisms. Vessel wall oxidases and oxygenases, in particular, are critical sources of oxygen radical production and can lead to an overall impairment of vascular ^aNO signaling, via the metalloprotein and free radical-mediated consumption of this vasoactive molecule. Vascular oxidase and oxygenase activities can thusaccount for the functional inactivation of ^aNO, leading to a prooxidative milieu and chronic inflammation.     

Protective role of endothelial nitric oxide synthase

Nitric oxide is a versatile molecule, with its actions ranging from haemodynamic regulation to anti-proliferative effects on vascular smooth muscle cells. Nitric oxide is produced by the nitric oxide synthases, endothelial NOS (eNOS), neural NOS (nNOS), and inducible NOS (iNOS). Constitutively expressed eNOS produces low concentrations of NO, which is necessary for a good endothelial function and integrity. Endothelial derived NO is often seen as a protective agent in a variety of diseases.This review will focus on the potential protective role of eNOS. We will discuss recent data derived from studies in eNOS knockout mice and other experimental models. Furthermore, the role of eNOS in human diseases is described and possible therapeutic intervention strategies will be discussed.     

一氧化氮、一氧化氮合酶和心脏功能

一氧化氮合酶(nitric　oxide synthase,NOS)催化L-精氨酸的氧化反应生成L-胍氨酸和一氧化氮(nitric oxide,NO)。其产物NO可通过依赖cGMP(环磷酸鸟苷)途径与非依赖cGMP途径发挥其复杂的生理学功能,如在心血管系统具有维持血管张力、调节血压,抑制血管平滑肌细胞迁移、增生,抑制血小板聚集与白细胞对血管壁的粘附以及调节影响心肌收缩与舒张功能的作用,并参与心率变异调节功能。本文就3种NOS同工酶的基因及其基因表达调节及影响因素进行简要综述。着重介绍nNOS1的心脏自主神经调节机制,iNOS对心脏收缩抑制以及心脏保护与损伤的双重作用,并对eNOS参与心功能调节的机制及其它生理、病理学等方面研究进展进行综述。     

Inhibitors caveolin-1 and protein kinase G show differential subcellular colocalization with Nitric oxide synthase

Background

Nitric oxide synthase (NOS) is negatively regulated by protein-protein interactions with caveolin-1 before extracellular activating signals release it for nitric oxide (NO) production. Smooth muscle protein kinase G (PKG) is a down-stream effector of NO signaling for relaxation of vascular smooth muscle cells (SMC). The PKG is also found in endothelial cells and it inhibits activated NOS within endothelial cells.

Methods

We used confocal fluorescence microscopy to colocalize the inhibitors caveolin-1 and PKG with NOS in freshly isolated neonatal lamb endothelial cells in order to corroborate the speculation of their differential effects on NOS. The roles of caveolin-1 and PKG as regulators of NOS were investigated by examining their respective subcellular sites of colocalization with NOS using qualitative fluorescence immunohistochemistry and confocal microscopy.

Results

Caveolin-1 was colocalized with NOS in the plasma membrane and Golgi. The PKG1-beta isoform was colocalized with serine116 phosphorylated NOS in the cytosol and in vesicular structures seen in the endoplasmic reticulum and in the nuclear region.

Conclusion

We conclude that unlike caveolin-1, a known pre-activation inhibitor of nascent NOS, PKG may be a post-activation inhibitor of NOS, possibly important for the recycling of the spent enzyme.     

Nitric oxide in the pathogenesis of vascular disease

Nitric oxide (NO) is synthesized by at least three distinct isoforms of NO synthase (NOS). Their substrate and cofactor requirements are very similar. All three isoforms have some implications, physiological or pathophysiological, in the cardiovascular system. The endothelial NOS III is physiologically important for vascular homeostasis, keeping the vasculature dilated, protecting the intima from platelet aggregates and leukocyte adhesion, and preventing smooth muscle proliferation. Central and peripheral neuronal NOS I may also contribute to blood pressure regulation. Vascular disease associated with hypercholesterolaemia, diabetes, and hypertension is characterized by endothelial dysfunction and reduced endothelium-mediated vasodilation. Oxidative stress and the inactivation of NO by superoxide anions play an important role in these disease states. Supplementation of the NOS substrate L-arginine can improve endothelial dysfunction in animals and man. Also, the addition of the NOS cofactor (6R)-5,6,7, 8-tetrahydrobiopterin improves endothelium-mediated vasodilation in certain disease states. In cerebrovascular stroke, neuronal NOS I and cytokine-inducible NOS II play a key role in neurodegeneration, whereas endothelial NOS III is important for maintaining cerebral blood flow and preventing neuronal injury. In sepsis, NOS II is induced in the vascular wall by bacterial endotoxin and/or cytokines. NOS II produces large amounts of NO, which is an important mediator of endotoxin-induced arteriolar vasodilatation, hypotension, and shock.     

MaxiK channel roles in blood vessel relaxations induced by endothelium-derived relaxing factors and their molecular mechanisms.

The endothelium of blood vessels plays a crucial role in the regulation of blood flow by controlling mechanical functions of underlying vascular smooth muscle. The regulation by the endothelium of vascular smooth muscle relaxation and contraction is mainly achieved via the release of vasoactive substances upon stimulation with neurohumoural substances and physical stimuli. Nitric oxide (NO) and prostaglandin I2 (prostacyclin, PGI2) are representative endothelium-derived chemicals that exhibit powerful blood vessel relaxation. NO action involves activation of soluble guanylyl cyclase and PGI2 action is initiated by the stimulation of a cell-surface receptor (IP receptor, IPR) that is coupled with Gs-protein-adenylyl cyclase cascade. Many studies on the mechanisms by which NO and PGI2 elicit blood vessel relaxation have highlighted a role of the large conductance, Ca2+-activated K+ (MaxiK, BKCa) channel in smooth muscle as their common downstream effector. Furthermore, their molecular mechanisms have been unravelled to include new routes different from the conventionally approved intracellular pathways. MaxiK channel might also serve as a target for endothelium-derived hyperpolarizing factor (EDHF), the non-NO, non-PGI2 endothelium-derived relaxing factor in some blood vessels. In this brief article, we review how MaxiK channel serves as an endothelium-vascular smooth muscle transducer to communicate the chemical signals generated in the endothelium to control blood vessel mechanical functions and discuss their molecular mechanisms.     

Plasma detection of NO by a catheter

Nitric oxide (NO) released by endothelial cells in response to hemodynamic shear stress is a key controller molecule of the vascular functions and antiatherogenic mechanisms. Endothelial dysfunction is associated with increased cardiovascular events. Therefore, several indirect techniques have been employed to evaluate endothelial function or NO bioavailability. However, a growing body of evidences suggests limitations of the indirect methods for evaluation of NO bioavailability. In years, it has been considered that NO is immediately oxidized or inactivated in blood stream. However, recent studies suggest that NO remain active in blood stream, causing remote biological response. Therefore, measuring plasma NO concentration directly in the circulation will contribute to clarify the kinetics and physiological roles of NO and to evaluate endothelial function. In this article, the measurement of plasma NO concentration using a newly developed catheter-type NO sensor will be described.     

The formation of an anti-restenotic/anti-thrombotic surface by immobilization of nitric oxide synthase on a metallic carrier

Coronary stenosis due to atherosclerosis, the primary cause of coronary artery disease, is generally treated by balloon dilatation and stent implantation, which can result in damage to the endothelial lining of blood vessels. This leads to the restenosis of the lumen as a consequence of migration and proliferation of smooth muscle cells (SMCs). Nitric oxide (NO), which is produced and secreted by vascular endothelial cells (ECs), is a central anti-inflammatory and anti-atherogenic player in the vasculature. The goal of the present study was to develop an enzymatically active surface capable of converting the prodrug l-arginine, to the active drug, NO, thus providing a targeted drug delivery interface. NO synthase (NOS) was chemically immobilized on the surface of a stainless steel carrier with preservation of its activity. The ability of this functionalized NO-producing surface to prevent or delay processes involved in restenosis and thrombus formation was tested. This surface was found to significantly promote EC adhesion and proliferation while inhibiting that of SMCs. Furthermore, platelet adherence to this surface was markedly inhibited. Beyond the application considered here, this approach can be implemented for the local conversion of any systemically administered prodrug to the active drug, using catalysts attached to the surface of the implant.     

Association of the eNOS E298D polymorphism and the risk of myocardial infarction in the Greek population

Background  

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), plays a key role in the regulation of vascular tone. Endothelium-derived NO exerts vasoprotective effects by suppressing platelet aggregation, leukocyte adhesion and smooth muscle cell proliferation. The E298D polymorphic variant of eNOS has been associated with myocardial infarction (MI), but data relating to this variant are divergent in Greece. Accordingly, we examined a possible association between the E298D polymorphism of the eNOS gene and MI in a subgroup of the Greek population.     

Nitric oxide producing coating mimicking endothelium function for multifunctional vascular stents

The continuous release of nitric oxide (NO) by the native endothelium of blood vessels plays a substantial role in the cardiovascular physiology, as it influences important pathways of cardiovascular homeostasis, inhibits vascular smooth muscle cell (VSMC) proliferation, inhibits platelet activation and aggregation, and prevents atherosclerosis. In this study, a NO-catalytic bioactive coating that mimics this endothelium functionality was presented as a hemocompatible coating with potential to improve the biocompatibility of vascular stents. The NO-catalytic bioactive coating was obtained by covalent conjugation of 3,3-diselenodipropionic acid (SeDPA) with glutathione peroxidase (GPx)-like catalytic activity to generate NO from S-nitrosothiols (RSNOs) via specific catalytic reaction. The SeDPA was immobilized to an amine bearing plasma polymerized allylamine (PPAam) surface (SeDPA-PPAam). It showed long-term and continuous ability to catalytically decompose endogenous RSNO and generate NO. The generated NO remarkably increased the cGMP synthesis both in platelets and human umbilical artery smooth muscle cells (HUASMCs). The surface exhibited a remarkable suppression of collagen-induced platelet activation and aggregation. It suppressed the adhesion, proliferation and migration of HUASMCs. Additionally, it was found that the NO catalytic surface significantly enhanced human umbilical vein endothelial cell (HUVEC) adhesion, proliferation and migration. The in vivo results indicated that the NO catalytic surface created a favorable microenvironment of competitive growth of HUVECs over HUASMCs for promoting re-endothelialization and reducing restenosis of stents in vivo.     

一氧化氮与脓毒性休克的关系

在脓毒性休克中,巨噬细胞和血管平滑肌细胞等细胞的诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达上调,使血管内皮源性舒张因子即一氧化氮(nitric oxide,NO)生成增多,引起血管过度扩张、微循环淤血、血管平滑肌对缩血管物质的反应性降低、心肌功能抑制等,导致严重的...     

一氧化氮合酶与主动脉瘤

一氧化氮合酶(NOS)通过催化L-Arg生成NO参与动脉瘤的变化。本文通过一氧化氮合酶在调节基质金属蛋白酶(MMPs)活性、调节平滑肌细胞凋亡以及动脉壁的氧化损伤中的作用对其与主动脉瘤形成的关系作一综述,若针对一氧化氮合酶在主动脉瘤形成中的关键环节进行干预,有可能为主动脉瘤的治疗提供新的途径。     

Neurogenic nitric oxide (NO) in the regulation of cerebroarterial tone

Sympathetic vasoconstrictor nerves are commonly recognized to mainly control the vascular smooth muscle tone, thus alters regional vascular resistance and blood flow. In contrast to peripheral organs and tissues, regulation by sympathetic nerves of blood flow in the brain is not so evident, and conversely vasodilator innervation is expected to play an important role. The mechanism underlying the neurogenic vasodilation in the cerebral artery has not been determined until recently. This problem was solved by the discovery of nitric oxide (NO) synthase inhibitors. Cerebral arterial dilatation caused by nerve stimulation is abolished by NO synthase inhibition and is restored by -arginine, a substrate of NO synthase; vasodilator nerve stimulation increases the production of cyclic GMP in the tissue and liberates NO_x (nitroxy compounds) from the arterial strip into superfusate. In addition, the presence of neurons containing NO synthase is histochemically demonstrated in the arterial wall. Neurogenic cerebral arterial dilation is thus hypothesized to be mediated by NO liberated as a neurotransmitter from the nerve. Nitroxidergic vasodilator innervation from the pterygopalatine ganglion would be important in the regulation of brain circulation.     

Nitric oxide and oxidative stress in vascular disease

Endothelium-derived nitric oxide (NO) is a paracrine factor that controls vascular tone, inhibits platelet function, prevents adhesion of leukocytes, and reduces proliferation of the intima. An enhanced inactivation and/or reduced synthesis of NO is seen in conjunction with risk factors for cardiovascular disease. This condition, referred to as endothelial dysfunction, can promote vasospasm, thrombosis, vascular inflammation, and proliferation of vascular smooth muscle cells. Vascular oxidative stress with an increased production of reactive oxygen species (ROS) contributes to mechanisms of vascular dysfunction. Oxidative stress is mainly caused by an imbalance between the activity of endogenous pro-oxidative enzymes (such as NADPH oxidase, xanthine oxidase, or the mitochondrial respiratory chain) and anti-oxidative enzymes (such as superoxide dismutase, glutathione peroxidase, heme oxygenase, thioredoxin peroxidase/peroxiredoxin, catalase, and paraoxonase) in favor of the former. Also, small molecular weight antioxidants may play a role in the defense against oxidative stress. Increased ROS concentrations reduce the amount of bioactive NO by chemical inactivation to form toxic peroxynitrite. Peroxynitrite—in turn—can “uncouple” endothelial NO synthase to become a dysfunctional superoxide-generating enzyme that contributes to vascular oxidative stress. Oxidative stress and endothelial dysfunction can promote atherogenesis. Therapeutically, drugs in clinical use such as ACE inhibitors, AT₁ receptor blockers, and statins have pleiotropic actions that can improve endothelial function. Also, dietary polyphenolic antioxidants can reduce oxidative stress, whereas clinical trials with antioxidant vitamins C and E failed to show an improved cardiovascular outcome.     

A New Hypothesis on the Pathogenesis of Intestinal Pseudo-obstructionby Intestinal Neuronal Dysplasia (IND)

Using morphometry and image analysis, we investigated 17 patients showing intestinal pseudo-obstruction secondary to intestinal neuronal dysplasia (IND) and 20 controls. In addition to an increase in the number and size of the ganglia and the ganglionic cells, we also noted a significant increase in NO synthase-containing ganglionic cells. We found that this enzyme, responsible for the synthesis of nitrous oxide caused by oxidation of L-argynine aminoacid, is a neurotransmitter able to induce smooth muscle relaxation by activating cyclic AMP. If the increase in NO synthase-producing ganglionic cells is responsible for the increase in nitrous oxide production, one can hypothesize that an overproduction of nitrous oxide plays a role in the pathogenesis of intestinal pseudo-obstruction secondary to neuronal dysplasia. As NO synthase can be blocked, as was demonstrated by giving L-methil-arginine or N-G-nitro-L-argynine, it might be possible to treat intestinal pseudo-obstruction caused by intestinal neuronal dysplasia at the pharmacological level.     

Increased smooth muscle actin expression from bone marrow stromal cells under retinoic acid treatment: an attempt for autologous blood vessel tissue engineering

Vascular replacement in vital organs is sometimes necessary for human life for example because of atherosclerosis. Blood vessel tissue engineering is applied for autologous transplantations to avoid graft rejections. Stem cells are used for blood vessel tissue engineering because they are the origin of smooth muscle cells, endothelial cells and fibroblasts. This paper shows that bone marrow stromal cells (BMSCs) can be induced to differentiate into the early stage of smooth muscle cells by using 0.01 microM retinoic acid. The differentiation of BMSCs to smooth muscle cells was detected by the expression of smooth muscle alpha actin (SM alpha-actin), the earliest smooth muscle cell marker. The SM alpha-actin marker expression was demonstrated using indirect immunofluorescence technique and Western blot analysis. The induction of BMSC to form early stages of smooth muscle cells in this study is appropriate for blood vessel tissue engineering because the early stage smooth muscle cells may be stimulated to develop vascular walls with endothelial cells using a co-culture system.     

Endothelin attenuates endothelium-dependent platelet inhibition in man

Aim: The vascular endothelium produces several substances, including nitric oxide (NO) and endothelin-1 (ET-1), which participate in the regulation of vascular tone in humans. Both these substances may exert other actions of importance for cardiovascular disease, e.g. effects on vascular smooth muscle cell proliferation and inflammation, and NO inhibits platelet function. Experiments were designed to investigate the effect of ET-1 on endothelium-dependent vasodilatation and attenuation of platelet activation. Methods: In 25 healthy male subjects (25 ± 1 years), forearm blood flow was measured by venous occlusion plethysmography, and platelet activity was assessed by whole blood flow cytometry (platelet fibrinogen binding and P-selectin expression) in unstimulated and adenosine diphosphate (ADP)-stimulated samples during administration of ET-1, the endothelium-dependent vasodilator acetylcholine and the NO synthase inhibitor l -NMMA. Results: Acetylcholine increased forearm blood flow and significantly inhibited platelet activation in both unstimulated and ADP-stimulated samples. In samples stimulated with 0.3 μm ADP, fibrinogen binding decreased from 41 ± 4% to 31 ± 3% (P < 0.01, n = 11) after acetylcholine administration. The vasodilator response to acetylcholine was significantly impaired during infusions of ET-1 and l -NMMA. ET-1 did not affect platelet activity per se, whereas l -NMMA increased platelet P-selectin expression. Both ET-1 and l -NMMA attenuated the acetylcholine-induced inhibition of platelet activity. Conclusions: Our study indicates that, further to inhibiting endothelium-dependent vasodilatation, ET-1 may also attenuate endothelium-dependent inhibition of platelet activation induced by acetylcholine. An enhanced ET-1 activity, as suggested in endothelial dysfunction, may affect endothelium-dependent platelet modulation and thereby have pathophysiological implications.     

Large arterioles in the control of blood flow: role of endothelium-dependent dilation

Although it is generally assumed that small arterioles form the major site of vascular resistance, microcirculatory studies revealed that 40-55% of the total network resistance can reside in large arterioles and small arteries. Thus, the mechanisms that control smooth muscle tone in these vessels have a major impact on the overall conductance of the vascular network. These control mechanisms are different from those in small arterioles: Aside from an apparently reduced sensitivity to metabolites, the large resistance vessels are normally too far away from the capillary areas which they feed to be reached by diffusing metabolites from dependent cells within a reasonable period of time. Rather, recent intravital microscopic studies suggest that large resistance vessels are under tight control of endothelial factors such as nitric oxide and endothelium-derived hyperpolarising factor (EDHF). Nitric oxide opposes myogenic constrictions of large arterioles that potentially would impair tissue perfusion and oxygenation. Moreover, nitric oxide and EDHF play an important role in the co-ordination of large and small resistance vessel behaviour that is pivotal for the adaptation of blood flow to altered tissue oxygen demands.     

Interstitial cells of Cajal are innervated by nitrergic nerves and express nitric oxide-sensitive guanylate cyclase in the guinea-pig gastrointestinal tract

Nitric oxide (NO) is a major signaling molecule in the gastrointestinal tract, and released NO inhibits muscular contraction. The actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC, NO-sensitive GC) and a subsequent increase in cGMP concentration. To elucidate NO targets in the gastrointestinal musculature, we investigated the immunohistochemical localization of the beta1 and alpha1 subunits of sGC and the distribution of neuronal NO synthase (nNOS) -containing nerves in the guinea-pig gastrointestinal tract. Distinct immunoreactivity for sGCbeta1 and sGCalpha1 was observed in the interstitial cells of Cajal (ICC), fibroblast-like cells (FLC) and enteric neurons in the musculature. Double immunohistochemistry using anti-c-Kit antibody and anti-sGCbeta1 antibody revealed sGCbeta1 immunoreactivity in almost all intramuscular ICC throughout the entire gastrointestinal tract. Immunoelectron microscopy revealed that sGCbeta1-immunopositive cells possessed some of the criteria for intramuscular ICC: presence of caveolae; frequently associated with nerve bundles; and close contact with smooth muscle cells. sGCbeta1-immunopositive ICC were closely apposed to nNOS-containing nerve fibers in the muscle layers. Immunohistochemical and immunoelectron microscopical observations revealed that FLC in the musculature also showed sGCbeta1 immunoreactivity. FLC were often associated with nNOS-immunopositive nerve fibers. In the myenteric layer, almost all myenteric ganglia contained nNOS-immunopositive nerve cells and were surrounded by myenteric ICC and FLC. Myenteric ICC in the large intestine and FLC in the entire gastrointestinal tract showed sGCbeta1 immunoreactivity in the myenteric layer. Smooth muscle cells in the stomach and colon showed weak sGCbeta1 immunoreactivity, and those in the muscularis mucosae and vasculature also showed evident immunoreactivity. These data suggest that ICC are primary targets for NO released from nNOS-containing enteric neurons, and that some NO signals are received by FLC and smooth muscle cells in the gastrointestinal tract.