Endothelium-derived Relaxing Factor Research Articles

Resveratrol restores lysophosphatidylcholine‐induced loss of endothelium‐dependent relaxation in rat aorta tissue coinciding with inhibition of extracellular‐signal‐regulated protein kinase activation

To investigate whether resveratrol could restore the lysophosphatidylcholine (LPC)-induced loss of endothelium-dependent relaxation in in vitro cultured rat aorta tissue, first the effect of resveratrol on the loss of EDR was examined in this preparation. The results showed that resveratrol effectively attenuated the inhibition of LPC (10 μM) on both endothelium-derived relaxing factor (EDRF) and endothelium-derived hyperpolarizing factor (EDHF) in a concentration-dependent manner (1, 10, 100 μM). In addition, resveratrol inhibited elevated K+-induced vascular contracture, but had no significant effects on ACh (1 μM)-induced endothelium-dependent relaxation (EDR). A similar tendency was also observed with PD 98059 (30 μM), a selective inhibitor of ERK. When the cells were exposed to LPC (20 μM) the mRNA expression of eNOS and COX-1 mRNA were down-regulated, followed by a local induction of iNOS and COX-2. Resveratrol and PD 98059 successfully reversed the effects of LPC on relative mRNA expressions. Both resveratrol and PD 98059 inhibited the activation of ERK induced by LPC. These findings demonstrate that resveratrol can restore the LPC-induced loss of EDR in rat aorta and protect the endothelium against LPC-induced injuries via the inhibition of the inflammation-like response.

Nitroxyl (HNO) as a Vasoprotective Signaling Molecule

Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO(•)), is rapidly emerging as a novel nitrogen oxide with distinct pharmacology and therapeutic advantages over its redox sibling. Whilst the cardioprotective effects of HNO in heart failure have been established, it is apparent that HNO may also confer a number of vasoprotective properties. Like NO(•), HNO induces vasodilatation, inhibits platelet aggregation, and limits vascular smooth muscle cell proliferation. In addition, HNO can be putatively generated within the vasculature, and recent evidence suggests it also serves as an endothelium-derived relaxing factor (EDRF). Significantly, HNO targets signaling pathways distinct from NO(•) with an ability to activate K(V) and K(ATP) channels in resistance arteries, cause coronary vasodilatation in part via release of calcitonin-gene related peptide (CGRP), and exhibits resistance to scavenging by superoxide and vascular tolerance development. As such, HNO synthesis and bioavailability may be preserved and/or enhanced during disease states, in particular those associated with oxidative stress. Moreover, it may compensate, in part, for a loss of NO(•) signaling. Here we explore the vasoprotective actions of HNO and discuss the therapeutic potential of HNO donors in the treatment of vascular dysfunction.

Glucose-6-Phosphate Dehydrogenase Is a Regulator of Vascular Smooth Muscle Contraction

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 μM), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca(2+) independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKCδ inhibitor rottlerin (10 μM) or by knocking down PKCδ expression using siRNA. Phorbol 12, 13-dibutyrate (10 μM), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca(2+)](i) and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca(2+)](i), thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKCδ-dependent manner and that G6PD modulates Ca(2+) entry and CA contractions evoked by membrane depolarization.

Role of Reactive Oxygen Species in Hyperadrenergic Hypertension

Background— Oxidative stress, an excessive production of reactive oxygen species (ROS) outstripping antioxidant defense mechanisms, occurs in cardiovascular pathologies, including hypertension. In the present study, we used biochemical, physiological, and pharmacological approaches to explore the role of derangements of catecholamines, ROS, and the endothelium-derived relaxing factor nitric oxide (NO • ) in the development of a hyperadrenergic model of hereditary hypertension: targeted ablation (knockout [KO]) of chromogranin A ( Chga ) in the mouse. Methods and Results— Homozygous (−/−) Chga gene knockout (KO) mice were compared with wild-type (WT, +/+) control mice. In the KO mouse, elevations of systolic and diastolic blood pressure were accompanied by not only elevated catecholamine (norepinephrine and epinephrine) concentrations but also increased ROS (H 2 O 2 ) and isoprostane (an index of lipid peroxidation), as well as depletion of NO • . Renal transcript analyses implicated changes in Nox1/2 , Xo/Xdh , and Sod1,2 mRNAs in ROS elevation by the KO state. KO alterations in blood pressure, catecholamines, H 2 O 2 , isoprostane, and NO • could be abrogated or even normalized (rescued) by either sympathetic outflow inhibition (with clonidine) or NADPH oxidase inhibition (with apocynin). In cultured renal podocytes, H 2 O 2 production was substantially augmented by epinephrine (probably through β 2 -adrenergic receptors) and modestly diminished by norepinephrine (probably through α 1 -adrenergic receptors). Conclusions— ROS appear to play a necessary role in the development of hyperadrenergic hypertension in this model, in a process mechanistically linking elevated blood pressure with catecholamine excess, renal transcriptional responses, ROS elevation, lipid peroxidation, and NO • depletion. Some of the changes appear to be dependent on transcription, whereas others are immediate. The cycle could be disrupted by inhibition of either sympathetic outflow or NADPH oxidase. Because common genetic variation at the human CHGA locus alters BP, the results have implications for antihypertensive treatment as well as prevention of target-organ consequences of the disease. The results document novel pathophysiological links between the adrenergic system and oxidative stress and suggest new strategies to probe the role and actions of ROS within this setting.

ChemInform Abstract: Endothelial Modulation of Agonist‐Induced Vasoconstriction in Mesenteric Microcirculation

AbstractReview: 28 refs.

New perspectives on vascular wall signaling: role of perivascular adipocytes and fibroblasts.

This communication represents personal perspectives of recent development in the newly evolved areas in vascular signaling mechanisms at the anatomical level of vascular walls from outside in, that is, from perivascular adventitial side to effectuate the control of vascular reactivity. Since half a century ago, the focus of interest in vascular biology has been confined primarily to the study of the excitation-contraction coupling of vascular smooth muscle (VSM) as well as neuroeffector mechanisms. During the past 3 decades, considerable advancement in the understanding of vascular signaling has been made via the discovery of endothelium-derived relaxation factors (EDRF), endothelium-derived hyperpolarizing factors (EDHF) and endothelium-derived contracting factors (EDCF). The discovery of nitric oxide (NO) as a major cellular messenger has also helped open up another huge area of research in oxidative stress and vascular diseases. In the past decade, concepts on vascular wall signaling have been extended from vascular endothelial cells and then translated to the other seemingly inert cellular components, such as perivascular adipocytes and adventitial fibroblasts. Growing body of evidences show that these cellularities contribute to both functional as well as structural integrity in vasculature with significant pathophysiological implications.

COX-mediated endothelium-dependent contractions: from the past to recent discoveries

Endothelial cells release various substances to control the tone of the underlying vascular smooth muscle. Nitric oxide (NO) is the best defined endothelium-derived relaxing factor (EDRF). Endothelial cells can also increase vascular tone by releasing endothelium-derived contracting factors (EDCF). The over-production of EDCF contributes to the endothelial dysfunctions which accompanies various vascular diseases. The present review summarizes and discusses the mechanisms leading to the release of EDCFs derived from the metabolism of arachidonic acid. This release can be triggered by agonists such as acetylcholine, adenosine nucleotides or by stretch. All these stimuli are able to induce calcium influx into the endothelial cells, an effect which can be mimicked by calcium ionophores. The augmentation in intracellular calcium ion concentration initiates the release of EDCF. Downstream processes include activation of phospholipase A(2) (PLA(2)), cyclooxygenases (COX) and the production of reactive oxygen species (ROS) and vasoconstrictor prostanoids (endoperoxides, prostacyclin, thromboxane A(2) and other prostaglandins) which subsequently diffuse to, and activate thromboxane-prostanoid (TP) receptors on the vascular smooth muscle cells leading to contraction.

Vascular nitric oxide: formation and function

Nitric oxide (NO) is a structurally simple, highly versatile molecule that was originally discovered over 30 years ago as an endothelium-derived relaxing factor. In addition to its vasorelaxing effects, NO is now recognized a key determinant of vascular health, exerting antiplatelet, antithrombotic, and anti-inflammatory properties within the vasculature. This short-lived molecule exerts its inhibitory effect on platelets largely through cGMP-dependent mechanisms, resulting in a multitude of molecular effects by which platelet activation and aggregation are prevented. The biosynthesis of NO occurs via the catalytic activity of nitric oxide synthase (NOS), an oxido-reductase found in many cell types. Nitric oxide insufficiency can be attributed to limited substrate/cofactor availability as well as interactions with reactive oxygen species (ROS). Impaired NO bioavailability represents the central feature of endothelial dysfunction, a common abnormality found in many vascular diseases. In this review, we present an overview of NO synthesis and biochemistry, discuss the mechanisms of action of NO in regulating platelet and endothelial function, and review the effects of vascular disease states on NO bioavailability.

Regeneration of the Endothelium in Vascular Injury

The endothelium mediates relaxations (dilatations) of the underlying vascular smooth muscle cells. The endothelium-dependent relaxations are due to the release of non-prostanoid vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also release substances (endothelium-derived hyperpolarizing factor, EDHF) that cause hyperpolarization of the cell membrane of the underlying vascular smooth muscle. The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive G(i) (alpha(2)-adrenergic activation, serotonin, thrombin) and insensitive G(q) (adenosine diphosphate, bradykinin) coupling proteins. The ability of the endothelial cell to release relaxing factors can be upregulated by impregnation with estrogens, exercise and antioxidants, and down-regulated by oxidative stress and increased presence of oxidized LDL. Following injury or apoptotic death, the endothelium regenerates. However, in regenerated endothelial cells, there is an early selective loss of the pertussis-toxin sensitive mechanisms of EDRF-release. Functional studies suggest that abnormal handling of LDL because of increased oxidative stress play a key role in this selective loss. Genomic analysis demonstrates the emergence of fatty acid binding protein-A (A-FBP) and metalloproteinase-7 (MMP7) in regenerated endothelial cells. The reduced release of NO resulting from the endothelial dysfunction in regenerated areas creates a locus minoris resistentiae which favors the occurrence of vasospasm and thrombosis as well as the initiation of atherosclerosis.

Obesity and aging: determinants of endothelial cell dysfunction and atherosclerosis

Endothelial cells are both the source and target of factors contributing to atherosclerosis. After the discovery of the endothelium-derived relaxing factor (EDRF) by Robert F. Furchgott in 1980 it soon became clear that endothelial cells also release vasoactive factors distinct from nitric oxide (NO) namely, endothelium-derived contracting factors (EDCF) as well as hyperpolarizing factors (EDHF). Vasoactive factors derived from endothelial cells include NO/EDRF, reactive oxygen species, endothelins and angiotensins which have either EDRF or EDCF functions, cyclooxygenase-derived EDCFs and EDRFs, and EDHFs. Endothelial factors are formed by enzymes such as NO synthase, cyclooxygenase, converting enyzmes, NADPH oxidases, and epoxigenases, among others, and participate in the regulation of vascular homeostasis under physiological conditions; however, their abnormal regulation due to endothelial cell dysfunction contributes to disease processes such as atherosclerosis, arterial hypertension, and renal disease. Because of recent changes in world demographics and the declining health status of the world's population, both aging and obesity as independent risk factors for atherosclerosis-related diseases such as coronary artery disease and stroke, will continue to increase in the years to come. Obesity and associated conditions such as arterial hypertension and diabetes are now also some of the primary health concerns among children and adolescents. The similarities of pathomechanisms activated in obesity and aging suggest that obesity--at least in the vasculature--can be considered to have effects consistent with accelerated, "premature" aging. Pathomechanisms as well as the clinical issues of obesity- and aging-associated vascular changes important for atherosclerosis development and prevention are discussed.

Diverse Mechanisms of Endothelium-Derived Hyperpolarizing Factor-Mediated Dilatation in Small Myometrial Arteries in Normal Human Pregnancy and Preeclampsia1

This ex vivo study focuses on the mechanisms of endothelium-dependent dilatation in the uterine circulation of normal pregnancy (n = 12) and in women with preeclampsia (n = 12). Arteries (internal diameter, ∼250 μm) isolated by myometrial biopsy from women undergoing planned cesarean delivery or delivery as a result of the deterioration of preeclampsia were studied using a wire myograph. Bradykinin-induced dilatation was assessed in the presence and/or absence of pharmacological inhibitors to determine the contribution of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF), as well as that of EDHF-mediated pathways such as myoendothelial gap junctions (MEGJs) and products of arachidonic acid, H(2)O(2) and cytochrome P450 2C9 (CYP2C9). Transmission electron microscopy was used to visualize morphological prerequisites for MEGJs. In normal pregnancy, EDHF through MEGJs appeared to be a predominant mediator conferring endothelium-dependent relaxation in small myometrial arteries. In preeclampsia, bradykinin-induced relaxation was reduced via compromised EDHF-type responses, in which the contribution of MEGJs became negligible. The attenuated role of MEGJs to endothelium-dependent relaxation was partly compensated through the contribution of H(2)O(2) or other endothelium-derived relaxing factors. CYP2C9 products of arachidonic acid had no effect on EDHF-type relaxation in arteries of women with normal pregnancy or with preeclampsia. We suggest that EDHF-type responses via MEGJs are primarily targeted in small myometrial arteries in women with preeclampsia. This could significantly contribute to the impaired uteroplacental blood flow in this disorder.

Stimuli of Sensory-Motor Nerves Terminate Arterial Contractile Effects of Endothelin-1 by CGRP and Dissociation of ET-1/ETA-Receptor Complexes

BackgroundEndothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism.Methodology/Principal findingsIn isolated rat mesenteric resistance arteries, ETA-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ETA-antagonism, were observed to promote dissociation of pre-existing ET-1/ETA-receptor complexes.ConclusionsIrreversible binding and activation of ETA-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1.

NO control: nitric oxide directly regulates substrate delivery to NOS. Focus on “Nitric oxide can acutely modulate its biosynthesis through a negative feedback mechanism on l-arginine transport in cardiac myocytes”

although the amino acid arginine is commonly associated with nitric oxide (NO) production via NO synthase (NOS), it also participates in the synthesis of urea, creatine, creatinine, agmatine, polyamines, as well as overall protein synthesis. Furthermore, it also influences hormone release (insulin,

Endothelial Modulation of Agonist-induced Vasoconstriction in Mesenteric Microcirculation

It is widely accepted that vascular endothelium regulates vasoconstriction via release of endothelium-derived relaxing factors (EDRF). The mesenteric circulation, which is the largest vascular bed, influences regulation of systemic blood pressure. However, the role of EDRF in the modulation of vascular tone in peripheral mesenteric circulation has not been extensively studied. Therefore, our recent studies investigated the role of the vascular endothelium in the regulation of methoxamine (alpha(1)-adrenoceptor agonist)-induced vasoconstriction and their age-related changes in rat mesenteric vascular beds. In mesenteric vascular beds with intact endothelium isolated from 8 week-old rats, the initial maximum vasoconstriction induced by continuous perfusion of methoxamine was time-dependently decreased during 3 hour-perfusion. Neither nitric oxide synthase inhibitor nor cyclooxygenase inhibitor altered this time-dependent reduction of methoxamine-induced vasoconstriction. Endothelium removal, K(+)-channel inhibitors and gap junction inhibitor significantly inhibited the time-dependent reduction of methoxamine-induced vasoconstriction. In the preparations with intact endothelium from 16 week-old rats, the time-dependent reduction of methoxamine-induced vasoconstriction disappeared. Furthermore, endothelium removal and treatment with cyclooxygenase inhibitor, thromboxane A(2) receptor antagonist or superoxide dismutase mimetic significantly reduced the methoxamine-induced vasoconstriction in the preparations from 16 week-old rats. These findings suggest that vascular endothelium acts to depress methoxamine-induced vasoconstriction by releasing endothelium-derived hyperpolarizing factor (EDHF), and dysfunction in this endothelial modulation develops with ageing.

Correction: Corrigendum: Kynurenine is an endothelium-derived relaxing factor produced during inflammation

Corrigendum: Kynurenine is an endothelium-derived relaxing factor produced during inflammation

4-Aminopyridine-sensitive K+ channels contributes to NaHS-induced membrane hyperpolarization and relaxation in the rat coronary artery

The present study aimed at examining the role of potassium channels and endothelium in relaxations induced by sodium hydrogen sulphide (NaHS), which is the donor of gaseous hydrogen sulphide (H2S) and the effect of NaHS on endothelium-dependent relaxations in rat coronary arteries. Rat coronary arteries were suspended in a myograph for force measurement and changes of the membrane potential in arteries were determined by membrane potential-sensitive fluorescence dye. NaHS relaxed coronary arteries pre-contracted by U46619 and the relaxation was significantly less in high KCl-contracted rings. NaHS-induced relaxations were reduced by 4-aminopyridine (4-AP) but unaffected by glibenclamide, iberiotoxin, NG-nitro-L-arginine methyl ester, ODQ, indomethacin or by endothelium removal. The inhibitory effect of 4-AP was absent in NaHS-induced relaxations in high KCl-contracted rings. Addition of NaHS caused membrane hyperpolarization and this effect was inhibited by 4-AP but not by glibenclamide. NaHS causes endothelium-independent relaxations in rat coronary arteries partially through activation of 4-AP-sensitive potassium channel and ensuring hyperpolarization. Other potassium channels, Na+–K+ pump or endothelium-derived relaxing factors play little role.

Thyroid hormone: the modulator of erectile function in the rabbit.

The possible role of thyroid hormones in the Nitric Oxide (NO)-mediated response to sexual stimulation, and on prostaglandin E1 (PGE1) and Sildenafil in the treatment of erectile dysfunction was investigated using the corpus cavernosum of the New Zealand rabbit animal model. The parameters studied were penile erection monitored as contractile force of the erectile tissue, sperm count and motility; in parallel with the haematocrit, red cell count or rheology, Heart Rate (HR), Mean Arterial Pressure (MAP), Thyroid Stimulating Hormones (TSH) and Thyroxine levels. Hypothyroidism or thyroidectomy was found to cause depletion of Endothelium Derived Relaxant Factor (EDRF) thereby causing very feeble contraction of the cavernosum muscle, in both prostaglandin E1 (PGE1) and sildenafil, oligospermia and less than 45 % motile sperms. Thyroxine treatment produced contraction proportionate to the concentrations of PGE1 and Sildenafil; providing evidence that the erectogenic actions of both PGE1 and Sildenafil are possible only in the presence of adequate thyroid hormone level.

A Pharmacogenetics‐based Approach to Reduce Cardiovascular Mortality with the Prophylactic Use of Statins

Nitric oxide (NO) is the main endothelial-derived relaxation factor and plays a major role in cardiovascular homeostasis. This key signalling molecule is synthesised by a family of nitric oxide synthases (NOS), and the endothelial isoform (eNOS) is the most important for nitric oxide formation in the cardiovascular system. Cardiovascular drugs including statins increase eNOS expression and up-regulate NO formation, and this effect may be responsible for protective, pleiotropic effects produced by statins. However, the genetic background may also affect NO formation in the cardiovascular system, and recent studies have shown that genetic polymorphisms in the eNOS gene modify endogenous NO formation and the risk of developing cardiovascular diseases. For example, cases with the CC genotype for the T(-786)C polymorphism in the eNOS gene are at increased cardiovascular risk when compared with those with the TT genotype. Interestingly, pharmacogenetic studies have recently indicated that atorvastatin improves NO formation more clearly in these individuals. However, it is not known whether this polymorphism really increases cardiovascular morbidity and mortality, and whether atorvastatin or other statins attenuate the morbidity and mortality rates in cases with the CC genotype. If proved true, then statins-induced up-regulation of eNOS and increased NO formation could compensate for a genetic 'disadvantage' in cases with the CC genotype. This could be a significant advance in the prevention of cardiovascular events. It is necessary although to validate this hypothesis with clinical trials which will require a long follow-up to assess relevant clinical events and not only surrogate biomarkers.

Human endothelial dysfunction: EDRFs

Since the detection of nitric oxide two and a half decades ago, there has been an incredible boost in endothelial function research, which is fascinating the research community. Physiologically, endothelial cells synthesize a number of vasoactive substances. In particular, several endothelium-derived relaxing factors (EDRFs) have been characterized, whereby nitric oxide is the most important. In humans, endothelial dysfunction is one of the first clinically detectable alterations in the development of atherosclerosis and is characterized by an imbalance in the release of vasoactive substances. Thus, it is the aim of this article to give an overview about endothelial function in humans, to summarize the different possibilities to assess endothelial function in this species, and to give an overview of the role of EDRFs in different cardiovascular diseases.

Hormonal modulation of endothelial NO production

Since the discovery of endothelium-derived relaxing factor and the subsequent identification of nitric oxide (NO) as the primary mediator of endothelium-dependent relaxations, research has focused on chemical and physical stimuli that modulate NO levels. Hormones represent a class of soluble, widely circulating chemical factors that impact production of NO both by rapid effects on the activity of endothelial nitric oxide synthase (eNOS) through phosphorylation of the enzyme and longer term modulation through changes in amount of eNOS protein. Hormones that increase NO production including estrogen, progesterone, insulin, and growth hormone do so through both of these common mechanisms. In contrast, some hormones, including glucocorticoids, progesterone, and prolactin, decrease NO bioavailability. Mechanisms involved include binding to repressor response elements on the eNOS gene, competing for co-regulators common to hormones with positive genomic actions, regulating eNOS co-factors, decreasing substrate for eNOS, and increasing production of oxygen-derived free radicals. Feedback regulation by the hormones themselves as well as the ability of NO to regulate hormonal release provides a second level of complexity that can also contribute to changes in NO levels. These effects on eNOS and changes in NO production may contribute to variability in risk factors, presentation of and treatment for cardiovascular disease associated with aging, pregnancy, stress, and metabolic disorders in men and women.