Nitric Oxide In Atherosclerosis Research Articles

Enhancement of Endothelial KCa Channel Activity as a Novel Strategy to Oppose Atherosclerosis

Atherosclerosis is a harmful cardiovascular condition in which low-density lipoprotein cholesterol (LDL-C) elevates in the blood, passes through the arterial endothelium, and promotes endothelial dysfunction (ED). ED can facilitate fatty plaque formation, which decreases intraluminal arterial diameter and blood flow, and increases arterial stiffness. A hallmark feature of ED is decreased bioavailability of nitric oxide (NO), a pluripotent compound with vasodilatory effects, along with anti-inflammatory and anti-proliferative actions in the arterial wall. Our group has previously reported that pharmacological activation of endothelial Ca2+-activated K+ channels, i.e. KCa2.3 and KCa3.1, can reverse ED, in part by enhancing endothelial NO production. Given the contributions of ED and NO in atherosclerosis, we hypothesize that enhancing endothelial function will mitigate the development and/or severity of atherosclerosis. The aim of this proof-of-concept study is to determine whether prolonged administration of the KCa channel activator SKA-31 in vivo can mitigate atherosclerosis by decreasing ED, arterial stiffness and/or fatty plaque formation. In this presentation, I will describe preliminary data obtained during the initial phase of this study. To mimic human atherosclerosis, we are using male Apoe knockout (Apoe-/-) mice, which begin to develop signs of progressive aortic injury at ~8 weeks of age. Experimentally, Apoe-/- mice on a regular chow diet receive one of three daily treatments for 16 weeks: SKA-31 (10 mg/kg), the KCa3.1 channel blocker senicapoc (40 mg/kg) or drug vehicle alone. As previous studies have reported that KCa3.1 channel blockers can reduce atherosclerotic events, we are using this treatment condition as a benchmark in our study. Drugs are formulated in miglyol and condensed milk, which is readily ingested by the mice. Plasma LDL-C levels are being measured to determine whether drug treatment modifies the levels of this important pathogenic molecule. Echocardiography is being used to measure standard cardiac function, along with aortic flow and pulse wave velocity as markers of arterial stiffness. Following euthanasia, vasodilatory responses of the aorta are examined by wire myography to determine whether drug treatment improves endothelial function. Histopathological analyses are being performed to elucidate aortic structure and integrity (with H&E staining) and the extent of atherosclerotic plaque formation in the aorta (with Oil Red O staining). Lastly, macrophage proliferation in the aortic wall will be assessed by immunocytochemistry, as macrophages proliferate rapidly in fatty plaque sites. Positive results from this study will facilitate future research to investigate the underlying cellular mechanisms by which endothelial KCa channel activation opposes atherogenesis.

Roles of Vascular Oxidative Stress and Nitric Oxide in the Pathogenesis of Atherosclerosis.

Major reactive oxygen species (ROS)-producing systems in vascular wall include NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase, xanthine oxidase, the mitochondrial electron transport chain, and uncoupled endothelial nitric oxide (NO) synthase. ROS at moderate concentrations have important signaling roles under physiological conditions. Excessive or sustained ROS production, however, when exceeding the available antioxidant defense systems, leads to oxidative stress. Animal studies have provided compelling evidence demonstrating the roles of vascular oxidative stress and NO in atherosclerosis. All established cardiovascular risk factors such as hypercholesterolemia, hypertension, diabetes mellitus, and smoking enhance ROS generation and decrease endothelial NO production. Key molecular events in atherogenesis such as oxidative modification of lipoproteins and phospholipids, endothelial cell activation, and macrophage infiltration/activation are facilitated by vascular oxidative stress and inhibited by endothelial NO. Atherosclerosis develops preferentially in vascular regions with disturbed blood flow (arches, branches, and bifurcations). The fact that these sites are associated with enhanced oxidative stress and reduced endothelial NO production is a further indication for the roles of ROS and NO in atherosclerosis. Therefore, prevention of vascular oxidative stress and improvement of endothelial NO production represent reasonable therapeutic strategies in addition to the treatment of established risk factors (hypercholesterolemia, hypertension, and diabetes mellitus).

Expression of nitric oxide related enzymes in coronary heart disease

Enzymes involved in the metabolism nitric oxide (NO) and reactive oxygen species (ROS) may play a role for the decreased availability of NO in atherosclerosis. We, therefore, hypothesized that the pattern of gene expression of these enzymes is altered in atherosclerosis. Myocardial tissue from patients with coronary heart disease (CHD) or without CHD (control group) was investigated. The level of enzymes related to NO/ROS metabolism was determined both at mRNA level and protein level by rt-PCR, real-time PCR, and western blot. The expression of NOS1-3 (synthesis of NO), arginase1 (reduction of L-arginine), p22phox (active subunit of NADPH oxidase), GTPCH (rate limiting enzyme for tetrahydrobiopterin), SOD1-3 (scavengers of superoxide anions), PRTMT1-3, and DDAH2 (involved in the metabolism of ADMA) was determined. All enzymes were found to be expressed in human myocardium. NOS isoforms were decreased in CHD in protein level, but only the downregulation of NOS3 expression reached statistical significance. The expression of PRMT1 and PRMT3 was increased. In addition, the expression of DDAH2 was reduced, both theoretically leading to an increase of ADMA concentration. SOD3 was downregulated in tissue from patients with CHD. Taken together, in myocardial tissue from patients with atherosclerosis, the expression of genes increasing ADMA levels is enhanced in contrast to a reduced expression of genes promoting NO synthesis. These results may contribute to the explanation of increased oxidative stress in atherosclerosis on the level of gene expression.

Nitric oxide, atherosclerosis and the clinical relevance of endothelial dysfunction.

The endothelium plays a key role in vascular homeostasis through the release of a variety of autocrine and paracrine substances, the best characterized being nitric oxide. A healthy endothelium acts to prevent atherosclerosis development and its complications through a complex and favorable effect on vasomotion, platelet and leukocyte adhesion and plaque stabilization. The assessment of endothelial function in humans has generally involved the description of vasomotor responses, but more widely includes physiological, biochemical and genetic markers that characterize the interaction of the endothelium with platelets, leukocytes and the coagulation system. Stable markers of inflammation such as high sensitivity C-reactive protein are indirect and potentially useful measures of endothelial function for example. Attenuation of the effect of nitric oxide accounts for the majority of what is described as endothelial dysfunction. This occurs in response to atherosclerosis or its risk factors. Much remains to be learned about the molecular and genetic pathophysiological mechanisms of endothelial cell abnormalities. However, pharmacological intervention with a growing list of medications can favorably modify endothelial function, paralleling beneficial effects on cardiovascular morbidity and mortality. In addition, several small studies have provided tantalizing evidence that measures of endothelial health might provide prognostic information about an individual patient's risk of subsequent events. As such, the sum of this evidence makes the clinical assessment of endothelial function an attractive surrogate marker of atherosclerosis disease activity. The review will focus on the role of nitric oxide in atherosclerosis and the clinical relevance of these findings.

Mechanisms of the pro- and anti-oxidant actions of nitric oxide in atherosclerosis.

The association of nitric oxide (NO) with cardiovascular disease has long been recognized and the extensive research on this topic has revealed both pro- and anti-atherosclerotic effects. While these contradictory findings were initially perplexing recent studies offer molecular mechanisms for the integration of these data in the context of our current understanding of the biochemistry of NO. The essential findings are that the biochemical properties of NO allow its exploitation as both a cell signaling molecule, through its interaction with redox centers in heme proteins, and an extremely rapid reaction with other biologically relevant free radicals. The direct reaction of NO with free radicals can have either pro- or antioxidant effects. In the cell, antioxidant properties of NO can be greatly amplified by the activation of signal transduction pathways that lead to the increased synthesis of endogenous antioxidants or down regulate responses to pro-inflammatory stimuli. These findings will be discussed in the context of atherosclerosis.

Nitric oxide synthase in atherosclerosis and vascular injury: insights from experimental gene therapy.

Gene therapy aims to intervene in a disease process by transfer and expression of specific genes in a target tissue or organ. Cardiovascular gene therapy in humans remains in its infancy, but in the last decade, experimental gene transfer has emerged as a powerful biological tool to investigate the function of specific genes in vascular disease pathobiology. Nitric oxide synthases, the enzymes that produce nitric oxide, have received considerable attention as potential candidates for vascular gene therapy because nitric oxide has pleiotropic antiatherogenic actions in the vessel wall, and abnormalities in nitric oxide biology are apparent very early in the atherogenic process. In this article, we review the use of nitric oxide synthases in experimental vascular gene therapy and assess the utility of these approaches for investigating the role of nitric oxide in atherosclerosis and their potential for human gene therapy.

Nitric oxide, nitrates and ischaemic preconditioning.

Time for primary review 24 days. In 1986 Furchgott first suggested that endothelial-derived relaxing factor might be nitric oxide (NO) [1]. Since then the remarkable role of nitric oxide (NO) as a modulator of biological phenomena has led to the question of its involvement within the spectrum of cardiovascular disease. The resulting research has implicated NO in atherosclerosis, hypertension, cardiomyopathy, pre-eclampsia, endotoxaemia [2] and cardiac allograft rejection [3] providing important additional insights into the pathogenesis of vascular and heart muscle disease. Although initially characterised in the vasculature NO is present in heart muscle [4] whilst the relative expression of the three isoforms (ncNOS, iNOS, ecNOS) responsible for its synthesis may have important implications in disease pathology. Apart from a cytotoxic role, NO has a role in cytostasis both regulating normal homeostasis and protecting against cell injury [5]. This, has led to the hypothesis that NO is cardioprotective in ischaemic heart disease. In testing this hypothesis very recent research has implicated NO in the phenomenon of ischaemic preconditioning. The current evidence suggesting its involvement in this exciting field is the focus of this review. Ischaemic preconditioning (PC) describes the profound myocardial protection that follows a short period of sublethal ischaemia and was recently considered by a NHBLI workshop to be the most powerful intervention to reduce myocardial infarct size other than reperfusion [6]. Initially described by Murray et al. in 1986 [7], in trying to understand the metabolic consequences of ischaemia, they found that brief periods of repetitive ischaemia in a canine model produced significant protection against myocardial cell death. There appears to be a bimodal distribution of protection with an initial more powerful but short-lived phase existing between 1 and 3 h (‘Early Preconditioning’) and a delayed less powerful phase occurring between 12 and 72 hours … * Corresponding author. Tel.: +44-171-922-8191; fax: +44-171-960-5659 m.marber{at}umds.ac.uk

Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide?

Atherosclerosis is a major contributor to cardiovascular disease, and genetic disorders of lipoprotein metabolism are recognized risk factors in atherogenesis. The gaseous monoxides nitric oxide (NO) and carbon monoxide (CO), generated within the blood vessel wall, have been identified as important cellular messengers involved in the regulation of vascular smooth muscle tone. Microsomal heme oxygenases degrade heme to biliverdin and CO, and the cytosolic enzyme biliverdin reductase then catalyzes reduction of biliverdin to bilirubin, both powerful chain-breaking antioxidants. Two principal isozymes of heme oxygenase have been identified, a constitutive isoform HO-2 (M(r) approximately 34,000) and an inducible isoform HO-1 (M(r) approximately 32,000), which is expressed at a low basal level in vascular endothelial and smooth muscle cells and is induced by heavy metals, oxidative stress, inflammatory mediators and oxidized low density lipoproteins. Although NO and CO modulate intracellular cGMP levels, platelet aggregation and smooth muscle relaxation, CO has a much lower affinity for soluble guanylyl cyclase than NO. Decreased production or sensitivity to NO in atherosclerosis may be compensated for by an induction of HO-1, with bilirubin acting as a cellular antioxidant and CO as a vasodilator. This review examines the evidence that oxidized low density lipoproteins (LDL), hypoxia and pro-inflammatory cytokines induce HO-1 expression and activity in vascular endothelial and smooth muscle cells, and evaluates the anti-atherogenic potential of the heme oxygenase signalling pathway.

The role of nitric oxide in atherosclerosis.

aCooke Pharma, Belmont bDivision of Cardiovascular Medicine, Stanford University, Stanford, California, USA.

The role of nitric oxide in atherosclerosis

The role of nitric oxide in atherosclerosis