The vasculature undergoes considerable stress

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1 Review Paper Vascular System: Role of Nitric Oxide in Cardiovascular Diseases Ka Bian, MD, PhD; 1 Marie-Françoise Doursout, PhD; 2 Ferid Murad, MD, PhD 1 In contrast with the short research history of the enzymatic synthesis of nitric oxide (NO), the introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in Glyceryl trinitrate (nitroglycerin) is the first compound of this category. On October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine or Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning NO as a signaling molecule in the cardiovascular system. NO-mediated signaling is a recognized component in various physiologic processes (eg, smooth muscle relaxation, inhibition of platelet and leukocyte aggregation, attenuation of vascular smooth muscle cell proliferation, neurotransmission, and immune defense), to name only a few. NO has also been implicated in the pathology of many inflammatory diseases, including arthritis, myocarditis, colitis, and nephritis and a large number of pathologic conditions such as amyotrophic lateral sclerosis, cancer, diabetes, and neurodegenerative diseases. Some of these processes (eg, smooth muscle relaxation, platelet aggregation, and neurotransmission) require only a brief production of NO at low nanomolar concentrations and are dependent From the Brown Institute of Molecular Medicine 1 and the Department of Anesthesiology, The University of Texas Health Science Center at Houston, 2 Houston, TX Address for correspondence: Ferid Murad, MD, PhD, The University of Texas Health Science Center at Houston, The Brown Institute of Molecular Medicine, 1825 Pressler Street, SRB- 530A, Houston, TX ferid.murad@uth.tmc.edu Manuscript received November 5, 2007; revised November 20, 2007; accepted December 10, ID: 6632 on the recruitment of cyclic guanosine monophosphate ()dependent signaling. Other processes are associated with direct interaction of NO or reactive nitrogen species derived from it with target proteins and requires a more sustained production of NO at higher concentrations but do not involve the pathway. J Clin Hypertens (Greenwich). 2008;10: Le Jacq The vasculature undergoes considerable stress and demands such as blood stream shear force effects and the constant generation of reactive oxygen and nitrogen species. A healthy vasculature depends on the integrity of the endothelium with a dynamic autocrine and paracrine organ with vasodilator, antiinflammatory, and antithrombotic effects. 1 Injuries or dysfunctional endothelial cells with a loss of endothelium-derived nitric oxide (NO) are major critical factors in the pathogenesis of vascular diseases, including hypertension, atherosclerosis, and vasospasm with compromised blood flow. Substantial knowledge of the NO signaling pathway has been gained during the past 3 decades. In 1977, Murad s laboratory reported that nitrovasodilators such as nitroglycerin and nitroprusside caused smooth muscle relaxation via generation of NO that activated soluble guanylyl cyclase and increased the concentration of cyclic guanosine monophosphate () in tissues 2 (Figure 1). NO s function as a possible messenger was first proposed in Furchgott and Zawadzki 3 further described an endothelium-derived relaxing factor identified as NO. In addition to its discovery as a vasodilator, NO mediates several protective functions of the endothelium by inhibiting (1) neutrophil activation and adhesion, (2) platelet adhesion and aggregation, (3) vascular smooth muscle proliferation, and (4) expression of proinflammatory 304

2 NH 2 OH NaN 3 H 2 O 2 NaNO 2 Phenylhydrazine Nitroglycerin Nitroprusside Nitrosoureas Nitrosamines Other nitrovasodilators Catalase Peroxidase Cytochromes? Others O 2 Oxidizing or reducing agents or spontaneous Inactive guanylate cyclase NO NO 2 Oxidizing agents (O 2, methylene blue, H 2 O 2, K 3 Fe(CN) 6 ) Hemoglobin Myoglobin Active guanylate cyclase Figure 1. Effects of nitrovasodilators and nitric oxide (NO) on soluble guanylyl cyclase. cytokines. Thus, a healthy endothelium with normal generation of NO acts to prevent atherosclerosis development and its complications. In contrast to the beneficial effects of NO at physiologic concentrations (nanomolar), large amounts (micromolar) of NO can be toxic and proinflammatory. For example, NO reacts with O 2 that can form peroxynitrous acid, a very unstable and reactive oxidizing species. Involvement of peroxynitrite in inflammatory diseases has been determined by detection of nitrotyrosine formation in various inflamed tissues 4,5 and is the most widely studied mechanism of protein nitration. 6 Nitrite (NO 2 ) is another major oxidation product derived from NO and can be oxidized by peroxidase/myeloperodixase to form reactive nitrogen intermediate(s), such as nitrogen dioxide that is capable of nitrating tyrosine. A distinct and important signaling role in mammalian biology of the anion, however, has recently been suggested. Physiologic NO 2 concentrations not only partially account for the benefit effect of NO, but also readily affect production, cytochrome P450 activities, heat shock protein 70, and heme oxygenase-1 expression in a variety of tissues, 7 thus NO 2 homeostasis may be of great importance to NO biology. NO-mediated signaling is a recognized component in various physiologic processes. Nevertheless, NO has also been implicated in the pathology of many inflammatory diseases, including arthritis, myocarditis, colitis, and nephritis and a large number of pathologic conditions such as amyotrophic lateral sclerosis, cancer, diabetes, and neurodegenerative diseases. Some of these processes (eg, smooth muscle relaxation, platelet aggregation, and neurotransmission) require only a brief production of NO at low nanomolar concentrations and are dependent on the recruitment of -dependent signaling. Other processes are associated with direct interaction of NO or reactive nitrogen species derived from it with target proteins and requires a more sustained production of NO at higher concentrations but do not involve the pathway. Through this article, a great deal of information will be provided in a way of relevance for medical practitioners. Endothelium-Centered Healthy Environment for Vasculature Since the study of Furchgott and Zawadzki, 3 which underlined the obligatory role of the endothelium in mediating acetylcholine-induced vasodilatation, VOL. 10 NO. 4 april 2008 THE Journal of Clinical Hypertension 305

3 PLATELET INHIBITORS ENDOTHELIUM- DEPENDENT VASODILATORS ACETYCHOLINE, HISTAMINE, THROMBIN, ATP, ETC. NITROVASODILATORS NO OONO - THROMBOXANE A2 SYNTHESIS ARGININE NO SOLUBLE GUANYLYL CYCLASE c GMP GTP EDRF CGMP Ca ++ ARGININE EDRF NO PARTICULATE GUANYLYL CYCLASE ANF-1 GTP SOLUBLE GUANYLYL CYCLASE CYCLIC GMP (ACTIVE) (ACTIVE) G.CYCLASE ATRIOPEPTINS PROTEIN P-PROTEIN Ca ++ ANF-1 ANF-2 PI3 kinase Ca ++ Ca ++ ANTI-AGGREGATION GpIIb/IIIa GTP ANF-2 PLC PI DG IP s P-MYOSIN LIGHT CHAIN CONTRACTION MYOSIN LIGHT CHAIN Ca ++ RELAXATION ENDOTHELIUM SMOOTH MUSCLE Figure 2. Effects of nitrovasodilators, endothelial-dependent vasodilatators, and atriopeptins on cyclic guanosine monophosphate () and vascular relaxation. GTP indicates guanosine-5-triphosphate; ANF, atrial natriuretic factor; EDRF, endothelium-derived relaxing factor; ONOO, peroxynitrite; NO, nitric oxide; PI3, phosphoinositide 3; Gp, glycoprotein; PLC, phospholipase; PI, phosphatidylinositol; DG, diacylglycerol; IP, inositol 3 or 4 phosphate. NO has been recognized as an endogenous nitrovasodilator that mediates the local regulation of basal arterial tone and is anticipated to vary in certain pathologic states. 8,9 For instance, Panza and colleagues 10 have indicated that patients with essential hypertension have a defect in the endothelium-derived NO system that may at least partly account for both the increased vascular resistance under basal conditions and the impaired response to endothelium-dependent vasodilators. Presently, our understanding of the process that leads to hypertension is allowing us to adopt principles of therapy that may be more beneficial for patients. Hypertension, particularly in highrisk patients, is a result of loss of balance and the absence of the ability to vasodilate normally. The interaction between the endothelial cell and the smooth muscle cell is very important in this process. The endothelium is a group of cells that produce compounds that are important in regulating vascular homeostasis by elaborating factors such as angiotensin II, NO, endothelin, and prostaglandins. Specifically, NO is found in endothelial cells responsible for smooth muscle relaxation. Gaseous NO diffuses across the endothelial cell and into the underlying smooth muscle cell, where it stimulates the pathway of guanylate cyclase to produce vasodilatation. 11 Normal endothelium maintains vascular tone and blood viscosity, prevents abnormal blood clotting and bleeding, limits inflammation of the vasculature, and suppresses smooth muscle cell proliferation. Abnormal endothelium causes increased inflammation and hypertrophy of the smooth muscle cells and promotes thrombosis and vasoconstriction, leading to the rapid growth of atherosclerotic plaques. Therefore, understanding endothelial vasculature will be imperative as researchers develop newer compounds that may enhance NO formation within the vasculature. NO Signaling and the Vascular System The introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in Glyceryl trinitrate (nitroglycerin [GTN]) is the first compound of this category and was synthesized by Sombrero in Another nitrate-containing compound, amyl nitrite, was discovered a few years later and was used by Guthrie and associates in During his discovery of dynamite, Alfred Nobel experienced angina pectoris and was prescribed nitroglycerin for chest pain in Almost a century later, 306

4 organic nitrates and their gaseous metabolic end product, NO, were implicated in a vast array of biologically diverse activities (Figure 2). Organic Nitrates Organic nitrates (RONO 2 ) are simple nitric and nitrous acid esters of alcohols. Clinically used, RONO 2 compounds include GTN, pentaerythrityltetranitrate, isosorbide dinitrate, and triethanolamine trinitrate biphosphate. Organic nitrates are susceptible to alkaline hydrolysis but are generally stable in neutral or weakly acidic solution, one of the major differences with NONOate compounds. Medical usage as well as the biological effects of organic nitrates can be attributed to their intracellular conversion to NO 2 ions and then to NO, 13 which, in turn, activates guanylate cyclase and increases the content in cells. 14,15 Elevated through activation of -dependent protein kinase (protein kinase G), protein phosphorylation, and regulation of cation channels, ultimately leads to dephosphorylation of the myosin light chain, resulting in smooth muscle relaxation. It has been suggested that multiple pathways contribute to NO formation from organic nitrates in vivo; however, the metabolic mechanism is poorly understood. Chen and colleagues 16 identified a nitrate reductase known as mitochondrial aldehyde dehydrogenase (mtaldh) that specifically converts GTN to 1,2-glyceryl dinitrate and NO 2. The NO 2 generated within the mitochondria is metabolized further to generate NO-based bioactivity by reduction to NO and/ or by conversion to S-nitrosothiol (SNO). These studies suggested that mechanism-based inactivation of mtaldh is involved in the development of GTN tolerance. Several medications such as chlorpropamide analogs, chloral hydrate, and acetaminophen can pharmacologically inhibit the activity of mtaldh. Thus, patients taking organic nitrates esters for the treatment of acute ischemic syndromes and congestive heart failure will benefit from awareness of certain drugs that inhibit mtaldh activity. S-nitrosothiols As early as 1981, Murad s and Ignarro s groups demonstrated that the bioactivities of certain pharmacologic nitrogen oxide donors were attributed to reactions with cellular thiols. 17 There is now a large body of literature that implicates SNO as an intermediate in NO-dependent and guanylyl cyclaseindependent signaling processes. SNO can be synthesized from the reaction between thiols and nitrous acid in extremely acidic conditions (ph <3). Reactive protein thiols are becoming regarded as major intracellular targets of NO Although RSNOs such as S-nitroso-N-acetylpenicillamine, N-acetyl-Snitrosopenicillaminyl-S-nitrosopenicillamine, and S-nitrosoglutathione are commercially available, none have been used therapeutically due to the unpredictable rate of decomposition in the body. In contrast, an increasing number of proteins have been found to undergo S-nitrosylation in vivo. The study of S-nitrosohemoglobin is an example through which the significance of the process may be understood. Hemoglobin (Hb) is a tetrameric red blood cell protein composed of two a- and two b-chains, each containing a heme prosthetic group. The ability of Hb to reversibly bind O 2 is governed by a cycle of allosteric transitions between the R (relaxed, high O 2 affinity) and the T (tense, low O 2 affinity) conformations. In addition to its well-recognized function of O 2 /CO 2 transport, the allosteric transition of Hb has been proposed to capture and deliver a third gas, NO. 18 Hb contains a highly conserved cysteine residue at position b93, which is reactive toward NO in the R state and relatively unreactive in the deoxygenated T state. The allosteric transition in Hb from T to R promotes the transfer of an NO group from Hb s hemes to its thiols, forming HbCysb93NO (SNO- Hb). SNO-Hb is capable of transferring the NO group to cysteins of other peptides and proteins. For example, albumin is an abundant circulating protein that may serve as an effective reservoir of NO in its nitrosated form. 19 The angiotensinconverting enzyme (ACE) inhibitor captopril is a reduced thiol that has been reported to act as an NO donor after being nitrosated. 20 Plasminogen activator is another popular medication with the ability to undergo nitrosation. 21 Reactive Oxygen/Nitrogen Species and Hypertension Arterial hypertension represents one of the most common conditions associated with endothelial dysfunction. For instance, a chronic inflammatory process associated with NO production may lead to oxidative damage of the vascular endothelium, decreasing its function. The reaction of superoxide (produced by all aerobic cells) with NO generates peroxynitrite, a potent oxidizing agent, that can also cause biological oxidative stress. Increases in reactive oxygen and/or nitrogen species following inflammatory challenge have been demonstrated in different models of organ failure 22 and hypertension. 23,24 VOL. 10 NO. 4 april 2008 THE Journal of Clinical Hypertension 307

5 Protein Nitration and Atherosclerosis Protein tyrosine nitration is a post-translational modification of proteins. Nitrated proteins are continuously produced in cells by reactive oxygen and nitrogen species even under normal physiologic conditions. 5 During periods of inflammation, protein nitration is significantly increased and may contribute to the inflammatory process or toxicity. It is well accepted that protein tyrosine nitration could be a biomarker to evaluate the extent and perhaps the cause of tissue damage in cardiovascular diseases 25 because nitrated protein tyrosines have been detected in atherosclerosis by immunohistochemistry. 26 Bian and Murad 27 have shown that tyrosine nitration can also diminish a protein s effectiveness as a substrate for tyrosine kinases. It is now well known that oxidized low-density lipoprotein (LDL) is of central importance in triggering atherosclerosis, as Leeuwenburgh and colleagues 28 proposed that reactive nitrogen intermediates promote LDL oxidation in atherosclerosis. Cardiovascular System: A Major Target for Protein Nitration The biological role of heme has been extensively reviewed. 29 The presence of heme and peroxides modulate vascular cell survival and oxidative stress adaptation, providing a possible mechanism for the evolution of atherosclerotic lesions. 30 We have tested the hypothesis that both free heme and iron play a key role in nitrotyrosine formation and further demonstrated that isolated heme and free metals are capable of tyrosine nitration in the presence of hydrogen peroxide and NO 2. Our data show that hemoprotein-rich tissues such as cardiac muscle are vulnerable to protein nitration in pathologic conditions characterized by the overproduction of H 2 O 2 and NO 2 or NO. 31 Therefore, our studies offer important mechanisms that underlie the heme/iron catalyzed nitration. Further clarification of the behavior of the heme (iron) and reaction with NO 2 /H 2 O 2 system is essential to the development of therapeutic methods that will focus on the prevention of oxidation and nitration. Cardiovascular Therapeutic Agents and NO Signaling b-blockers b-blockers have historically been considered an effective and safe option for the initial treatment of hypertension. Evidence suggests that b-blockers work at the vascular biology level to produce NO release. b-blockers differ in terms of their b receptor selectivity, intrinsic sympathomimetic activity, and benefit/risk in diabetes and insulin sensitivity. Nebivolol, the newest of the b-blockers, is longacting and the most cardioselective b 1 -blocker currently available. 32 Nebivolol-induced endothelium-dependent vasodilation associated with activation of the l-arginine/no pathway may confer benefits to patients. The combined mechanisms of b-adrenoceptor antagonism and NO-mediated vasodilation may potentiate the blood pressure lowering effect of this agent and confer a broader favorable metabolic profile, which may be clinically relevant for hypertensive patients. The antioxidant effects of nebivolol and its neutral or even favorable effects on both carbohydrate and lipid metabolism are well documented. 33 These effects consistently differentiate nebivolol from nonvasodilating b-blockers such as atenolol, metoprolol, and bisoprolol. The risk for diabetes is lower, the metabolic effects are lower, and persons with diabetes who have clear NO dysfunction may have particular benefits from this agent. Therefore, third-generation b-blockers, such as labetolol, carvedilol, bucindolol, and nebivolol, vasodilate by different mechanisms, behaving differently than traditional b-blockers and offering different benefits. Compared with conventional b-blockers, vasodilating b-blockers have beneficial hemodynamic effects including decreased pressure wave reflection from the periphery, leading to decreases in central aortic blood pressure. Larger trials are needed, however, to determine whether reduced central pressure will translate into improved cardiovascular outcomes compared with nonvasodilating b-blockers. 34 ACE Inhibitors or AT 1 Receptor Blockers Several studies have shown that hypertension is associated with enhanced LDL oxidation and oxidative stress in the bloodstream and arterial tissue. 35 The reduction of exceeding oxidative stress after intervention with antihypertensive drugs is achieved in experimental models 35,36 and in hypertensive patients. 37 Among antihypertensive drugs, sulfhydryl ACE inhibitors reduce LDL oxidation, formation of oxidation-specific epitopes in the arterial wall, and atherogenic lesion formation in experimental models. The increased level of oxidant stress (ie, generation of oxygen radicals such as superoxide anion) is involved in the expression of hypertension in genetic and experimental (eg, angiotensin IIinduced) forms of hypertension. 38 This increased oxidant stress promotes oxidation of cysteine residues (ie, enhanced disulfide bond 308

6 formation) in functional proteins. The increased activity of the renin-angiotensin system plays a role in the expression of hypertension. It is likely that increased activity of the renin-angiotensin system elicits hypertension by generating oxidant stress since ACE inhibitors or angiotensin AT 1 receptor blockers, normalize blood pressure and endothelium-dependent vasodilation in hypertensive models, including spontaneously hypertensive rats 39 and reduce oxidant stress in these rats thereby improving the redox status of cysteine residues in proteins in cells and on cell membranes. The oxidative stress in patients with essential hypertension is improved by chronic administration of therapeutic doses of the sulfhydryl ACE inhibitor zofenopril but not with the nonsulfhydryl ACE inhibitor enalapril. This effect was coupled with an improvement of the NO/asymmetric dimethyl-l-arginine balance. Sulfhydryl ACE inhibition induces sustained reduction of systemic oxidative stress and improves the NO pathway in patients with essential hypertension. 40 In addition to exerting various beneficial actions on vascular structure and function beyond their blood pressurelowering effects, data from experimental studies show that ACE inhibitors can attenuate the development of atherosclerosis in a wide range of species. The postulated mechanisms of this atheroprotective effect are the antioxidant actions of ACE inhibitors and their enhancement of the endothelial elaboration of bioactive NO. Using bovine aortic endothelial cells in culture, Scribner and colleagues 40 assessed the comparative effects of 3 ACE inhibitors on endothelial NO production and action and endothelial oxidative stress. Results showed that the sulfhydryl ACE inhibitor zofenopril has unique effects compared with captopril and enalapril. This compound improves NO production and bioactivity and does so in conjunction with decreased endothelial cell oxidant stress. The biochemical basis for this protective mechanism is not entirely clear; however, these actions suggest that zofenopril may reduce endothelial effects of risk factors for atherothrombotic disease. Conclusions NO research has expanded rapidly in the past 20 years, and the role of NO in physiology and pathology have been extensively studied. The pathways of NO synthesis, signaling, and metabolism in vascular biological systems have been a major area of research resulting in the 1998 Nobel Prize in Medicine or Physiology. As a gas and free radical with an unshared electron, NO participates in various biological processes. The rapid growth of NO research has generated more than 80,000 publications in the field of NO signaling. The interaction between NO and proteins may be roughly divided into 2 categories. In many instances, NO mediates its biological effects by activating guanylyl cyclase and increases intracellular synthesis from guanosine-5 -triphosphate. The list of -independent effects of NO, however, is also growing at a rapid rate. In this review, a brief history of the medical usage of NO is introduced. The importance and relevance of overproduction of NO in cardiovascular pathology has been stressed. The utilization of intact cell cultures, tissues, and cell-free preparations with the use of pharmacologic, biochemical, and molecular biological approaches to characterize, purify, and reconstitute these NO regulatory pathways should lead to the development of new therapies for various pathologic conditions characterized by an unbalanced production of NO. Disclosures: Financial support of our research was provided by the John S. Dunn Foundation, Harold and Leila Mathers, the Robert A. 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