Oxidative stress and endothelial nitric oxide bioactivity

The endothelium plays an important role in the maintenance of vascular homeostasis. Central to this role is the endothelial production of nitric oxide (NO), synthesized by the constitutively expressed endothelial isoform of nitric oxide synthase. Vascular diseases, including hypertension, diabetes, and atherosclerosis, are characterized by impaired endothelium-derived NO bioactivity that may contribute to clinical cardiovascular events. Growing evidence indicates that impaired endothelium-derived NO bioactivity is due, in part, to excess vascular oxidative stress. This review outlines how different forms of oxidative stress can impact on NO bioactivity and discusses strategies to prevent oxidative stress-induced endothelial dysfunction.     

Endothelial function and oxidative stress.

Increased oxidative stress impairs endothelial function and is thought to mediate vascular disease. Several pathological conditions increase the production of reactive oxygen species (ROS) in the vascular wall, including hypercholesterolemia, diabetes, and hypertension. These conditions are associated with endothelial dysfunction and cardiovascular disease. Thus, overall vascular function is dependent upon the balance of oxidant and antioxidant mechanisms, which determines endothelial function. Endothelial function is usually defined as nitric oxide (NO) production and/or bioavailability. Because ROS can interact and inactivate NO, vascular oxidative stress can lead to decrease NO bioavailability. This results in endothelial dysfunction and increased risk of cardiovascular diseases. Several pharmacological approaches have been used to improve endothelial function and decrease oxidative stress. These include treatment modalities that augment the antioxidant defense mechanisms, increase NO production, and inhibit ROS-generating enzymes. This review provides an overview of the relationship between endothelial function and oxidative stress.     

Gene and stem cell therapy in the treatment of erectile dysfunction and pulmonary hypertension; potential treatments for the common problem of endothelial dysfunction

The endothelium has an important regulatory role in the maintenance of vascular homeostasis, vascular tone, blood flow, and in preserving a non-thrombogenic blood-tissue interface. Injury to the vascular wall with subsequent endothelial dysfunction alters these important regulatory functions leading to a state of abnormal endothelial function. In this paper, we review the pathophysiology of endothelial dysfunction and how this disorder is common to the development of erectile dysfunction and of pulmonary arterial hypertension. Current medical therapies for these two disorders are discussed followed by a review of the preclinical studies involving currently available strategies for gene and stem cell therapy and their potential for the clinical treatment of these two disorders of endothelial dysfunction.     

The role of the anti-ageing protein Klotho in vascular physiology and pathophysiology

Klotho is an anti-ageing protein that functions in many pathways that govern ageing, like regulation of phosphate homeostasis, insulin signaling, and Wnt signaling. Klotho expression levels and levels in blood decline during ageing. The vascular phenotype of Klotho deficiency features medial calcification, intima hyperplasia, endothelial dysfunction, arterial stiffening, hypertension, and impaired angiogenesis and vasculogenesis, with characteristics similar to aged human arteries.Klotho-deficient phenotypes can be prevented and rescued by Klotho gene expression or protein supplementation. High phosphate levels are likely to be directly pathogenic and are a prerequisite for medial calcification, but more important determinants are pathways that regulate cellular senescence, suggesting that deficiency of Klotho renders cells susceptible to phosphate toxicity. Overexpression of Klotho is shown to ameliorate medial calcification, endothelial dysfunction, and hypertension.Endogenous vascular Klotho expression is a controversial subject and, currently, no compelling evidence exists that supports the existence of vascular membrane-bound Klotho expression, as expressed in kidney. In vitro, Klotho has been shown to decrease oxidative stress and apoptosis in both SMCs and ECs, to reduce SMC calcification, to maintain the contractile SMC phenotype, and to prevent μ-calpain overactivation in ECs.Klotho has many protective effects with regard to the vasculature and constitutes a very promising therapeutic target. The purpose of this review is to explore the etiology of the vascular phenotype of Klotho deficiency and the therapeutic potential of Klotho in vascular disease.     

A study of magnesium deficiency in human and experimental pulmonary hypertension

Pulmonary hypertension (PH) is defined as an increase in mean pulmonary arterial pressure above 25?mmHg. Pulmonary vasoconstriction, cellular proliferation, inflammation, and oxidative stress are involved in the pathophysiology of PH. Since hypomagnesemia was reported to promote endothelial cell dysfunction leading to inflammation and oxidative stress, we investigated the potential involvement of magnesium (Mg) deficiency in experimental and human PH. Our results indicate that Mg deficiency has no impact on hypoxia-induced PH development or severity, and that no reduction in Mg plasma concentration was observed in patients with severe pulmonary arterial hypertension. Thus, hypomagnesemia does not appear to play a role in the pathophysiology of experimental and human pulmonary hypertension.     

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.     

Redox control of vascular nitric oxide bioavailability

NO is an important component of vascular homeostasis and abnormal NO bioactivity has been implicated in number of disease states with important public health implications. One clear mechanism of impaired NO bioactivity and vascular disease is excess vascular oxidative stress. There is now a wealth of developing data that manipulation of vascular antioxidant stress is the considerable influence of the biologic activity of endothelium-derived NO. It remains to be seen if this influence can be exploited in a manner that truly alters the course of human disease.     

Possible role of nitric oxide in the pathogenesis of pulmonary hypertension in broilers: a synopsis.

Nitric oxide (NO) produced by vascular endothelial cells is an important determinant of the basal tone of small arteries and arterioles. Impaired endothelial NO production has been implicated in the pathophysiology of pulmonary hypertension in humans. Available data suggest that reduction of endothelial NO synthesis, with evidence of reduced endothelial NO synthase expression in pulmonary arterioles, is associated with increased pulmonary vasomotor tone and vascular remodelling in hypertensive broilers. Supplemental l-arginine, a precursor of NO, has been shown to induce flow-dependent pulmonary vasodilation, to prevent reduced endothelial NO synthase expression and to inhibit vascular remodelling in broilers with pulmonary hypertension. Nevertheless, its effect on pulmonary hypertension syndrome incidence is limited. It appears that impaired production of NO is a secondary rather than a causative factor in the pathogenesis of pulmonary hypertension in broilers.     

Hyperketonemia (ketosis), oxidative stress and type 1 diabetes.

The long-term complications of diabetes are the leading causes of morbidity and mortality in the type 1 diabetic population and remain a major public health issue. Hyperglycemia is one of the major risk factors in the development of vascular complications. A growing body of evidence indicates that hyperglycemia leads to increased oxidative stress and monocyte and endothelial cell dysfunction. In addition to hyperglycemia, type 1 diabetic patients frequently experience ketosis (hyperketonemia). The blood concentration of ketone bodies reaches higher than 25mM in diabetics with severe ketosis. Traditionally, clinical practice has considered hypertketonemia to be present only in type 1 diabetic patients. Newer data indicate that diabetic ketoaciosis or hyperketonemia co-exists with hyperglycemia among older type 2 diabetic patients and in African Americans and other minority groups with type 2 diabetes. This review will focus on the role of hyperketonemia in the etiology of oxidative stress in diabetic patients. The data presented here illustrate that the ketone body acetoacetate (AA) can generate superoxide radicals and cause increases in oxidative stress and cellular dysfunction. The data included in this review demonstrate that blood levels of markers of oxidative stress are elevated in hyperketonemic patients compared with those of normoketonemic diabetic patients. Thus, both in vitro and in vivo research indicate that ketosis can generate oxygen radicals and result in excess cellular oxidative stress in type 1 diabetic patients. Elevated oxidative stress levels in ketotic patients can play a significant role in the development of vascular inflammation and contribute to the increased incidence of vascular disease and complications associated with type 1 diabetes.     

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.     

一氧化氮生物利用度失调与动脉粥样硬化

内皮功能障碍与动脉粥样硬化(AS)密切相关。氧化应激、脂质浸润、炎性反应因子表达及血管张力改变等涉及一氧化氮(NO)生物利用度(内源性NO的生成和利用)的降低,在内皮功能障碍中发挥重要作用。精氨酸酶活性增强、非对称性二甲基精氨酸和同型半胱氨酸增加均能使NO生物利用度下降,促进AS发生发展。糖尿病、肥胖、慢性肾病和吸烟等通过多种方式影响NO生物利用度,参与AS的发生。     

Nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension

Endothelial dysfunction in the setting of cardiovascular risk factors such as hypercholesterolemia, diabetes mellitus, chronic smoking, as well hypertension, is, at least in part, dependent of the production of reactive oxygen species (ROS) and the subsequent decrease in vascular bioavailability of nitric oxide (NO). ROS-producing enzymes involved in increased oxidative stress within vascular tissue include NADPH oxidase, xanthine oxidase, and mitochondrial superoxide producing enzymes. Superoxide produced by the NADPH oxidase may react with NO, thereby stimulating the production of the NO/superoxide reaction product peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, therefore switching an antiatherosclerotic NO producing enzyme to an enzyme that may accelerate the atherosclerotic process by producing superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and also occurs within the smooth muscle cell layer. Increased superoxide production has important consequences with respect to signaling by the soluble guanylate cyclase and the cGMP-dependent kinase I, which activity and expression is regulated in a redox-sensitive fashion. The present review will summarize current concepts concerning eNOS uncoupling, with special focus on the role of tetrahydrobiopterin in mediating eNOS uncoupling.     

Vascular nitric oxide and oxidative stress: determinants of endothelial adaptations to cardiovascular disease and to physical activity.

Cardiovascular disease is the single leading cause of death and morbidity for Canadians. A universal feature of cardiovascular disease is dysfunction of the vascular endothelium, thus disrupting control of vasodilation, tissue perfusion, hemostasis, and thrombosis. Nitric oxide bioavailability, crucial for maintaining vascular endothelial health and function, depends on the processes controlling synthesis and destruction of nitric oxide as well as on the sensitivity of target tissue to nitric oxide. Evidence supports a major contribution by oxidative stress-induced destruction of nitric oxide to the endothelial dysfunction that accompanies a number of cardiovascular disease states including hypertension, diabetes, chronic heart failure, and atherosclerosis. Regular physical activity (exercise training) reduces cardiovascular disease risk. Numerous studies support the hypothesis that exercise training improves vascular endothelial function, especially when it has been impaired by preexisting risk factors. Evidence is emerging to support a role for improved nitric oxide bioavailability with training as a result of enhanced synthesis and reduced oxidative stress-mediated destruction. Molecular targets sensitive to the exercise training effect include the endothelial nitric oxide synthase and the antioxidant enzyme superoxide dismutase. However, many fundamental details of the cellular and molecular mechanisms linking exercise to altered molecular and functional endothelial phenotypes have yet to be discovered. The working hypothesis is that some of the cellular mechanisms contributing to endothelial dysfunction in cardiovascular disease can be targeted and reversed by signals associated with regular increases in physical activity. The capacity for exercise training to regulate vascular endothelial function, nitric oxide bioavailability, and oxidative stress is an example of how lifestyle can complement medicine and pharmacology in the prevention and management of cardiovascular disease.     

Endothelial dysfunction and vascular disease – a 30th anniversary update

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best‐characterized endothelium‐derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium‐dependent hyperpolarizations, EDH‐mediated responses). As regards the latter, hydrogen peroxide (H₂O₂) now appears to play a dominant role. Endothelium‐dependent relaxations involve both pertussis toxin‐sensitive G_i (e.g. responses to α₂‐adrenergic agonists, serotonin, and thrombin) and pertussis toxin‐insensitive G_q (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low‐density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin‐sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H₂O₂), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium‐derived contracting factors. Recent evidence confirms that most endothelium‐dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium‐dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium‐dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin‐1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.     

TRPV1 activation prevents high-salt diet-induced nocturnal hypertension in mice

High dietary salt-caused hypertension is associated with increasing reactive oxygen species generation and reduced nitric oxide (NO) bioavailability. Transient receptor potential vanilloid type 1 (TRPV1), a specific receptor for capsaicin, is proposed to be involved in Dahl salt-sensitive hypertension, as determined in acute or short-term experiments. However, it remains unknown whether activation of TRPV1 by dietary capsaicin could prevent the vascular oxidative stress and hypertension induced by a high-salt diet. Here, we report that consumption of a high-salt diet blunted endothelium-dependent relaxation in mesenteric resistance arteries and elevated nocturnal blood pressure in mice. These effects were associated with increased superoxide anion generation and reduced NO levels in mesenteric vessels in mice on a high-salt diet. However, chronic administration of capsaicin reduced the high-salt diet-induced endothelial dysfunction and nocturnal hypertension in part by preventing the generation of superoxide anions and NO reduction of mesenteric arteries through vascular TRPV1 activation. Our findings provide new insights into the role of TRPV1 channels in the long-term regulation of blood pressure in response to high-salt intake. TRPV1 activation through chronic dietary capsaicin may represent a promising lifestyle intervention in populations with salt-sensitive hypertension.     

A computational model for free radicals transport in the microcirculation

Nitric oxide (NO), superoxide (O(2)(-)), and peroxynitrite (ONOO(-)) interactions in pathophysiologic conditions such as cardiovascular disease, hypertension, and diabetes have been studied extensively in vivo and in vitro. A reduction in bioavailability of NO is a common event that is known as the endothelial dysfunction in these conditions. Despite intense investigation of NO biotransport and O(2)(-) and ONOO(-) biochemical interactions in vasculature, we have very little quantitative knowledge of distributions and concentrations of NO, O(2)(-), and ONOO(-) under normal physiologic and pathophysiologic conditions. Based on fundamental principles of mass balance, vessel geometry, and reaction kinetics, we developed a mathematical model of these free radicals transport in and around an arteriole during oxidative stress. We investigated the role of O(2)(-) and ONOO(-) in inactivating vasoactive NO. The model predictions include (a) NO interactions with oxygen, O(2)(-), and ONOO(-) have relatively little effect on the NO level in the vascular smooth muscle under physiologic conditions; (b) superoxide diffuses only a few microns from its source, whereas peroxynitrite diffuses over a larger distance; and (c) reduced superoxide dismutase levels significantly increase O(2)(-) and peroxynitrite concentrations and decrease NO concentration. Model results indicate that the reduced NO bioavailability and enhanced peroxynitrite formation may vary depending on the location of oxidative stress in the microcirculation, which occurs at diverse vascular cell locations in diabetes, aging, and cardiovascular diseases. The results will have significant implications for our understanding of these free radical interactions in physiologic and pathophysiologic conditions resulting from endothelial dysfunction.     

Mechanisms of cardiovascular remodeling in hyperhomocysteinemia

In hypertension, an increase in arterial wall thickness and loss of elasticity over time result in an increase in pulse wave velocity, a direct measure of arterial stiffness. This change is reflected in gradual fragmentation and loss of elastin fibers and accumulation of stiffer collagen fibers in the media that occurs independently of atherosclerosis. Similar results are seen with an elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), which increases vascular thickness, elastin fragmentation, and arterial blood pressure. Studies from our laboratory have demonstrated a decrease in elasticity and an increase in pulse wave velocity in HHcy cystathionine β synthase heterozygote knockout (CBS(-/+)) mice. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-TIMP (tissue inhibitor of metalloproteinase) inhibitory tertiary complex. We have demonstrated the contribution of the NO synthase (NOS) isoforms, endothelial NOS and inducible NOS, in the activation of latent MMP. The differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP resulting in vascular remodeling in response to HHcy. The contribution of the NOS isoforms, endothelial and inducible in the collagen/elastin switch, has been demonstrated. We have showed that an increase in inducible NOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance. In addition, increased levels of Hcy compete and suppress the γ-amino butyric acid-receptor, N-methyl-d-aspartame-receptor, and peroxisome proliferator-activated receptor. The HHcy causes oxidative stress by generating nitrotyrosine, activating the latent MMPs and decreasing the endothelial NO concentration. The HHcy causes elastinolysis and decrease elastic complicance of the vessel wall. The treatment with γ-amino butyric acid-receptor agonist (muscimol), N-methyl-d-aspartame-receptor agonist (MK-801), and peroxisome proliferator-activated receptor agonists (ciprofibrate and ciglitazone) mitigates the cardiovascular dysfunction in HHcy.     

Thioredoxins, mitochondria, and hypertension

Endothelial dysfunction, often demonstrated by the loss of the endothelial cell's ability to cause vasodilation in response to appropriate stimuli, is one of the earliest events in the development of atherosclerosis. This has led to intense investigation of the factors affecting both the production and the degradation of NO, the endothelium-derived relaxing factor and a primary mediator of endothelial function. Reactive oxygen species (ROS), particularly superoxide anion, are well known to inhibit NO, and therefore the mechanisms by which endothelium regulates production of ROS are also of high interest. In this issue of The American Journal of Pathology, Zhang et al( 1) demonstrate regulation of such events by a mitochondria-specific thioredoxin, which reduces oxidative stress and increases NO bioavailability, thus preserving vascular endothelial cell function and preventing atherosclerosis development.     

NOS 3 subcellular localization in the regulation of nitric oxide production

Endothelium-derived nitric oxide (NO) is a key signalling molecule in the maintenance of cardiovascular health. Endothelial NO synthase (NOS 3), which catalyses the formation of NO, is targeted to the plasma membrane by dual acylation. In vitro studies suggest that membrane localization of NOS 3 is an important regulatory element of NO production. Dysfunction of the vascular endothelium and a decrease in NO bioavailability is associated with the development and progression of a number of cardiovascular diseases, including hypertension. Our laboratory has previously published that in salt-dependent hypertension there is an altered localization of NOS 3, with an increase in cytosolic expression. These data have led us to question whether the increased cytosolic NOS 3 expression is a form of compensation for endothelial dysfunction in hypertension, or an indicator and contributing factor to endothelial dysfunction. This review will outline the importance of subcellular localization in the regulation of NOS 3 in vitro, the role of NOS 3 in endothelial dysfunction associated with salt-dependent hypertension, and the potential physiological consequences of altered NOS 3 localization in vivo.