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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1873.)
© 2000 American Heart Association, Inc.

Brief Reviews

Nitric Oxide Synthase in Atherosclerosis and Vascular Injury

Insights From Experimental Gene Therapy

Keith M. Channon; HuSheng Qian; Samuel E. George

From the Department of Cardiovascular Medicine (K.M.C.), University of Oxford, John Radcliffe Hospital, Oxford, England, and the Division of Cardiology (H.Q., S.E.G.), Duke University Medical Center, Durham, NC.

Correspondence to Keith M. Channon, MD, MRCP, Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK OX3 9DU. E-mail keith.channon{at}cardiov.ox.ac.uk

	Abstract

Top Abstract NO and Atherogenesis Biological Effects of NOS... eNOS, nNOS, or iNOS? Adjunctive Strategies in NOS... Summary References

 
Abstract—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.

Key Words: nitric oxide • atherosclerosis • gene transfer • endothelium • adenovirus

	NO and Atherogenesis

Top Abstract NO and Atherogenesis Biological Effects of NOS... eNOS, nNOS, or iNOS? Adjunctive Strategies in NOS... Summary References

 
NO Synthases
Nitric oxide (NO) is produced by NO synthases (NOs), which oxidize L-arginine to L-citrulline (reviewed in References 1 and 2^{1 2} ). All 3 NOS isoforms have a similar molecular structure and require multiple cofactors, including flavins, NADPH, and tetrahydrobiopterin, that are required to maintain dimerization and NO production^{1 2} (the Figure). Neuronal (nNOS, or NOS I) and endothelial (eNOS, or NOS III) isoforms are constitutively expressed and are activated by calcium-calmodulin. The inducible isoforms (iNOS, or NOS II) are regulated primarily at the transcriptional level, independent of agonist stimulation and intracellular calcium levels.^{3 4} eNOS, expressed in endothelial cells, is the predominant NOS isoform in the vessel wall. Under basal conditions, eNOS is inactive and remains membrane bound by virtue of myristoylation, palmitoylation, and an inhibitory interaction with caveolin, the principal structural protein in caveolae. Receptor-mediated agonist stimulation (eg, bradykinin, substance P) leads to rapid enzyme activation by depalmitoylation, binding of calcium-calmodulin, displacement of caveolin, and release from the plasma membrane.^{5 6} Shear stress is an important physiological stimulator of eNOS activity, causing rapid membrane release and activation by Akt-dependent serine phosphorylation^{7 8} and upregulating eNOS gene expression by transcriptional activation of the eNOS promoter.⁹ After vessel injury or in disease states, iNOS expression may be induced in the media,¹⁰ atherosclerotic plaque,¹¹ or neointima.^{4 12} In normal blood vessels, nNOS is present in neurons in the adventitia and may be expressed by medial smooth muscle cells (SMCs) under pathophysiological conditions. Recent data also suggest that nNOS and its splice variant µNOS may be expressed at low levels in the media and adventitia.¹³

View larger version (19K): [in this window] [in a new window]   Figure 1. eNOS regulation and NO-superoxide interactions in atherosclerosis. The eNOS enzyme is a homodimer; each monomer has a reductase domain, incorporating binding sites for flavin cofactors (FAD and FMN) and for NADPH. The reductase domain supplies electrons to the oxidase domain of the other monomer, where L-arginine is oxidized at the active site, which incorporates a heme group (Fe). Electron transfer is controlled by the binding of calcium-calmodulin (CaM) to a region between the reductase and oxidase domains. Tetrahydrobiopterin (BH4) is required for homodimerization and modulates the heme redox state during electron transfer. Tetrahydrobiopterin is synthesized in cells from GTP by a multistep pathway; the rate-limiting first step is catalyzed by GTP–cyclohydrolase I. eNOS is activated by agonists that bind specific G protein–coupled receptors (eg, bradykinin [BK]), leading to elevations in intracellular calcium and activation of calmodulin binding. These receptors are concentrated in specialized plasma membrane domains, caveolae, with other signaling molecules. Caveolin, the major structural protein of caveolae, binds eNOS in an inactive state. Caveolin and calmodulin binding are exclusive; activation releases eNOS from the caveolae. Shear stress activates eNOS by phosphorylation at serine 1179 (P) through a phosphatidylinositol 3-kinase– dependent pathway, independent of calcium-calmodulin. NO may be scavenged by superoxide (OO), resulting in the production of peroxynitrite (OONO). SOD enzymes remove superoxide and generate hydrogen peroxide (H2O2).

Functions of Vascular NO
NO produced in the endothelium rapidly diffuses to interact with molecular targets in cells in the vascular wall and lumen.¹⁴ NO interacts with thiol groups and with metal centers in diverse protein targets, including membrane receptors, G proteins, ion channels, cytosolic enzymes, and transcription factors such as activator protein-1 and nuclear factor-B (reviewed in Reference 15¹⁵ ). S-Nitrosylation of thiol groups in plasma proteins such as albumin generates a circulating "pool" of NO-donating groups,¹⁶ whereas S-nitrosylation of hemoglobin in the lung provides nitrosothiol groups to the peripheral vasculature and regulates oxygen delivery.^{17 18}

In the vascular wall, NO activates soluble guanylate cyclase in vascular smooth muscle cells (VSMCs), leading to elevation of cGMP, activation of cGMP-dependent protein kinase (PKG), and vasorelaxation, the primary basis for blood flow and pressure regulation.¹⁹ In addition to regulating vascular tone, a substantial body of evidence suggests that NO has important antiatherogenic effects^{20 21} : (1) antiplatelet effects. NO inhibits platelet adhesion and aggregation²² and thrombin-induced expression of platelet-activating factor. (2) antiproliferative effects. NO donors potently inhibit VSMC proliferation,^{23 24} migration,²⁵ and extracellular matrix synthesis.²⁶ (3) anti-inflammatory effects. Atherogenic stimuli stimulate endothelial expression of adhesion molecules and chemokines, leading to inflammatory cell recruitment.²⁷ NO donors inhibit activation and nuclear translocation of nuclear factor-B,^{28 29} block cytokine-stimulated endothelial adhesion molecule expression,²⁹ and reduce adhesion and activation of neutrophils and monocytes.³⁰ In particular, inhibition of monocyte chemotactic protein-1 expression appears to be an important aspect of NO’s anti-inflammatory effect in atherosclerosis.^{31 32} (4) antioxidant effects. Unmodified LDL undergoes oxidative modifications in the vessel wall that render it highly atherogenic.³³ Continuous, low-level NO production may directly inhibit lipid oxidation by scavenging free radicals.³⁴

Rationale for NOS Gene Therapy in Vascular Disease
The observation of deficient NO-mediated vasorelaxation in hypercholesterolemia suggests that loss of NO bioactivity is an early feature of atherosclerosis.^{35 36 37} In animal models, treatment with NOS inhibitors potentiates neointimal proliferation,^{38 39} whereas supplemental L-arginine^{36 40} or NO adducts^{24 41 42} improve endothelial function and limit neointimal proliferation. Mice with targeted deletion of the eNOS gene are hypertensive⁴³ and respond to vascular injury with increased intimal hyperplasia and abnormal remodeling.^{44 45 46} NO also appears to play a significant role in human atherogenesis. Atherosclerosis is associated with reduced endothelial NO production,⁴⁷ and in patients with coronary artery disease, even angiographically normal coronary segments show paradoxical constriction to acetylcholine.⁴⁸ Endothelial function is also deficient in preatherosclerotic conditions such as hypercholesterolemia,^{49 50} diabetes mellitus,⁵¹ hypertension,⁵² smoking,^{53 54} and normal aging.⁴⁹ Treatment of hypercholesterolemia,⁵⁵ dietary supplementation with L-arginine,^{54 56 57} or antioxidant therapy⁵⁸ improves the functional deficit. These studies suggest that loss of normal NO bioavailability precedes progression to more advanced lesions.

NOS gene therapy aims to increase or restore vascular NO production in these NO deficiency states. NOS protein is absent or reduced in the endothelium overlying severe atherosclerotic lesions in arteries^{11 47} or diseased venous bypass grafts,⁵⁹ providing an immediate rationale for gene transfer to restore NOS levels in diseased endothelium or when the endothelium is lost, eg, after balloon injury. Atherogenic stimuli such as oxidized LDL may also reduce eNOS levels by inhibiting eNOS gene expression.⁶⁰ However, in other vascular disease states in which NO bioactivity is reduced, endothelial NOS protein levels appear to be maintained or even increased,^{61 62 63} suggesting that greater complexity underlies the observed reductions in NO bioactivity. Some potential mechanisms are discussed below.

Interactions of NO With Superoxide
Increased superoxide production may account for a significant proportion of the NO deficit in atherosclerotic vessels.⁶⁴ Superoxide reacts rapidly with NO, thereby reducing NO bioactivity and producing peroxynitrite,^{65 66} a strong oxidant that can have physiologically protective effects⁶⁷ but that at high levels causes tissue damage by nitrosylation of cellular proteins and lipids.⁶⁸ In addition, superoxide stimulates mitogenesis in VSMCs and activates other redox-sensitive signaling pathways.^{69 70} Increased superoxide production, principally by NAD(P)H oxidases and xanthine oxidase, accounts for a significant proportion of the NO deficit in several models of vascular disease, including hypercholesterolemia,^{71 72} atherosclerosis,^{72 73} hypertension,^{62 74 75} and heart failure.⁶¹ Correction of hypercholesterolemia reduces superoxide production and restores endothelium-dependent vasorelaxation.⁷⁶ Antioxidants⁷⁷ and superoxide dismutase (SOD)^{66 78 79} produce similar effects, further supporting the hypothesis that superoxide plays a role in the observed NO deficits.

NOS Dysregulation in Atherosclerosis
In addition to variations in eNOS protein levels and the interaction of NO with superoxide, regulation of the eNOS enzyme appears to be abnormal in vascular disease states, resulting in reduced NO production despite the presence of NOS protein. Furthermore, eNOS may generate superoxide when endothelial cells are subjected to stimuli such as hyperglycemia or oxidized LDL particles.^{80 81 82 83} This "dysregulation" of eNOS may result from several potential mechanisms.

The eNOS–caveolin 1 interaction maintains eNOS in an inactive, membrane-bound state,^{5 6 84} and is modulated by cholesterol and superoxide anions.⁸⁵ In endothelial cells subjected to hypercholesterolemia or oxidized LDL, eNOS activation by calcium-calmodulin is impaired, possibly due to increased caveolin expression, increased levels of caveolin-eNOS inhibitory complexes,⁸⁶ or disruption of normal caveolar lipid composition.⁸⁷ In contrast to its inhibitory interaction with caveolin, activated eNOS in the cytosol is stabilized by interaction with heat shock protein 90,⁸⁸ which tends to increase eNOS activity in response to calcium-mobilizing agonists and to shear stress–induced phosphorylation.^{7 8 89} Whether these or other eNOS regulatory mechanisms are also deranged in atherosclerosis remains to be investigated.

Tetrahydrobiopterin is a required cofactor for NOS activity: all 3 NOS isoforms require tetrahydrobiopterin for NOS homodimerization and for electron transfer during arginine oxidation.¹ Production of NO by endothelial cells requires adequate tetrahydrobiopterin,⁹⁰ and NOS synthesized in the absence of tetrahydrobiopterin is inactive for NO generation; exogenous tetrahydrobiopterin restores enzyme activity.⁹¹ More importantly, tetrahydrobiopterin-deficient NOS generates superoxide rather than NO.⁹² Recent data suggest that tetrahydrobiopterin levels are lower in the diabetic rat aorta and underlie the observed reduction in NO production.⁹³ Exogenous tetrahydrobiopterin partially restores endothelial NO bioactivity and reduces NOS-dependent superoxide production in hypertension,^{62 94} hypercholesterolemia,⁹⁵ and smokers,⁹⁶ whereas inhibition of tetrahydrobiopterin synthesis impairs NO-mediated vasorelaxation.⁹⁷ Thus, tetrahydrobiopterin may be an important factor in modulating NOS activity in vascular disease states.

Arginine availability and the relative concentration of endogenous NOS inhibitors such as asymmetric dimethylarginine^{98 99} can limit NO production, despite the presence of normal or high levels of NOS enzyme. High asymmetric dimethylarginine levels are associated with impaired NO-mediated vasorelaxations in hypercholesterolemia,⁹⁹ provide a rationale for the beneficial effects of arginine supplementation in vascular disease models,¹⁰⁰ and may have significant effects on NO production in vascular disease.¹⁰¹

These and other factors causing NOS dysregulation have important implications for NOS gene therapy, because simply augmenting NOS protein levels under these conditions may not increase NO bioactivity and could increase superoxide production, resulting in detrimental rather than beneficial effects. Experimental studies of NOS gene therapy need to determine whether recombinant NOS activity is accompanied by increased superoxide production and peroxynitrite formation and to establish the optimal level of NOS expression to achieve an appropriate increase in NO production without potentially detrimental effects.

	Biological Effects of NOS Vascular Gene Transfer

Top Abstract NO and Atherogenesis Biological Effects of NOS... eNOS, nNOS, or iNOS? Adjunctive Strategies in NOS... Summary References

 
Gene transfer vectors allow targeted expression of genes in cells in the vessel wall, provide tools to investigate the function of specific genes in disease processes, and may potentially lead to gene therapy strategies for vascular disease.¹⁰² For NOS gene transfer, adenovirus vectors have been most widely used, with a smaller number of studies using high-efficiency plasmid-liposome gene delivery and incorporating coat proteins from hemagglutinating virus of Japan. Whereas adenovirus results in the highest efficiency of gene transfer, liposome-mediated gene transfer is a virus-free system with little host immune response and potentially longer transgene expression. Because NO diffuses from NOS-targeted cells to surrounding cells (a "bystander" effect), very high-efficiency transduction may not be an absolute requirement to exert a biological effect. Experimental studies of NOS gene transfer initially evaluated the expression and activity of recombinant NOS isoforms in cultured cells,¹⁰³ then in isolated vascular rings after ex vivo infection and culture,^{104 105 106 107 108 109 110 111} and finally in several in vivo models of experimental gene therapy^{112 113 114 115 116 117 118 119 120 121 122 123 124 125} (the Table).

View this table: [in this window] [in a new window]   Table 1. Studies of Experimental Vascular Gene Therapy With NOS

Vasomotor Function
The ability to restore endothelial vasomotor function in dysfunctional arteries represents a critically important test of NOS gene transfer. Ex vivo eNOS gene transfer to isolated vascular rings from normal or hypercholesterolemic rabbit carotid arteries or aorta,¹⁰⁶ canine basilar arteries,¹⁰⁸ porcine coronary arteries,¹⁰⁷ or human saphenous veins¹¹⁰ improves vascular relaxations to a variety of vasoactive agonists, such as calcium ionophore, acetylcholine, or UTP. In vivo gene delivery of eNOS or nNOS confirms that NOS gene transfer can restore or augment regulated NO-mediated vasorelaxation in several settings, including normal,^{117 119 120} atherosclerotic,¹¹⁷ balloon-injured,¹¹³ or eNOS-deficient⁴⁵ arteries. Moreover, eNOS gene transfer reduces the vasoconstrictive response to hypoxia in rat lungs¹²⁴ and restores NO-mediated endothelial function in models of cardiac failure,¹²⁵ hypertension,^{126 127} or diabetes,¹²⁸ demonstrating that the functional effects of NOS gene transfer are not limited to pharmacological agonist stimulation ex vivo, nor to specific models of endothelial dysfunction such as hypercholesterolemia. Activation of recombinant NOS by shear stress, a major activator of eNOS in the normal vessel, has not been specifically studied after in vivo vascular gene transfer. However, hemodynamic data from the mouse lung after in vivo eNOS gene transfer suggest that recombinant eNOS is activated by increasing pulmonary blood flow, and it reduces pulmonary vascular resistance.¹²⁹ Taken together, these studies provide important landmarks in vascular gene transfer. They demonstrate that NOS gene transfer can be used to restore or augment NOS activity in the vessel wall and can modify physiologically regulated vascular function in both normal arteries and those in a variety of pathological states.

Intimal Hyperplasia
NO donors or compounds that increase intracellular cGMP inhibit VSMC proliferation,²³ VSMC migration,²⁵ and collagen synthesis,^{26 130} all key components of intimal hyperplasia. Exogenous NO-donating compounds such as molsidomine²⁴ or L-arginine¹³¹ also reduce intimal hyperplasia in vascular injury models in vivo. These results suggest that endothelial NO may limit VSMC proliferation and neointimal formation in vivo. Gene transfer studies provide direct evidence that local augmentation of vascular NO production by NOS isoforms inhibits intimal hyperplasia in several models of vascular injury: balloon-injured rat^{113 114 115} or pig¹¹⁶ arteries, rabbit or canine venous bypass grafts,^{123 132} and rat aortic allografts.¹²² This effect seems most likely due to direct inhibition of VSMC proliferation, as evidenced by bromodeoxyuridine or proliferating cell nuclear antigen staining^{112 132} and mediated by modulation of VSMC cell phenotype¹¹⁴ or cell cycle inhibition.^{133 134} In addition, NOS gene transfer inhibits VSMC migration and matrix remodeling.¹³⁵ Alternative mechanisms include increased VSMC apoptosis,¹³⁶ reduced inflammation or platelet activation,¹³⁷ or promotion of endothelial regrowth. These gene transfer studies strongly suggest a direct role for NO in modulating VSMC biology in the vessel wall, in response to disease states or vascular injury.

Regression of Atherosclerosis
Local gene transfer of NOS provides a powerful tool to address the importance and mechanisms underlying the "antiatherogenic" actions of NO in the vessel wall. In cholesterol-fed rabbits, nNOS gene transfer reduces endothelial adhesion molecule expression, restores endothelium-dependent vasomotor relaxation toward normal, and rapidly reduces T-lymphocyte and monocyte infiltration.¹¹⁸ These findings indicate an important role for NO in regulating endothelial cell activation and inflammatory cell infiltration in early atherosclerosis. The striking rapid changes in monocyte/macrophage infiltration suggest that foam cell formation may be a highly dynamic process in early experimental atherosclerosis. In particular, an important mediator of the antiatherogenic effect of NOS gene transfer in vivo appears to be downregulation of vascular monocyte chemotactic protein-1 expression (D. Citrin and S.E. George, unpublished data, 1999),^{31 32} as previously demonstrated in cultured endothelial cells after eNOS gene delivery.¹³⁸

	eNOS, nNOS, or iNOS?

Top Abstract NO and Atherogenesis Biological Effects of NOS... eNOS, nNOS, or iNOS? Adjunctive Strategies in NOS... Summary References

 
There is no a priori reason to suppose that one particular NOS isoform will necessarily be superior for gene therapy in vascular disease. All have been shown to increase vascular NO production, and all may have equivalent promise for vascular gene transfer. However, functional differences between the isoforms may affect NOS regulation after gene transfer, and further studies need to address these potential differences to make a rational choice for NOS gene therapy.

Many investigators have chosen eNOS as a candidate for vascular gene transfer on the basis that eNOS is the predominant isoform present in the normal vessel wall.^{112 113 116} eNOS gene transfer increases vascular NO production in normal and diseased arteries, whether directed at the endothelial surface or at adventitial fibroblasts.^{121 139} In balloon-injured arteries, eNOS is expressed in medial SMCs and increases basal cGMP levels.¹²⁴ Thus, cells other than endothelial cells are able to support eNOS activity and therefore, represent targets for vascular NOS gene therapy. In adventitial fibroblasts, recombinant eNOS appears to be localized in caveolae and is regulated by membrane-Golgi trafficking in response to bradykinin stimulation.¹²¹ This approach may be an alternative to luminal delivery when the endothelium is disrupted or diseased^{111 139} and also provides novel routes for vascular gene delivery, eg, to the adventitia of cerebral arteries via the cerebrospinal fluid.^{121 140 141} Adventitial eNOS expression augments ex vivo vasomotor responses stimulated by pharmacological agonists in the absence of an endothelium and also increases basal cGMP levels in vivo, even though adventitial fibroblasts are not exposed to shear stress.¹²¹ Nevertheless, future studies need to determine how adventitial eNOS is activated in blood vessels in vivo and whether adventitial eNOS can modify vascular injury or disease states in a similar manner to luminal eNOS gene delivery.

Increasing evidence suggests that iNOS expression is increased in medial SMCs, in the neointima after vascular injury,^{10 142 143} or in allografts after transplantation,¹²² suggesting that iNOS expression might limit the thrombotic and proliferative response in injured vessels.⁴ If so, iNOS may be particularly suited to gene transfer in vascular injury states. In vivo studies in balloon-injured arteries¹¹⁵ and in aortic allografts¹²² show that iNOS gene transfer generates high-level NO production, independent of agonist stimulation or shear stress. Sustained NO production, with the higher V_max of the iNOS enzyme, may be an advantage over other isoforms, but concern remains that continuous high activity could also increase superoxide production, leading to peroxynitrite formation.¹⁴⁴

nNOS is abundant in neurons and skeletal muscle and appears to be expressed in medial SMCs and in the adventitia under normal and pathological conditions.^{11 13} Like eNOS, nNOS is expressed constitutively and regulated by calcium-calmodulin, but it has a higher V_max for NO production. We have used nNOS as a candidate for vascular gene transfer^{117 118} and have shown that nNOS acts as a functional surrogate for eNOS in normal or atherosclerotic arteries^{117 118} and in vein grafts¹³² and that it is activated by agonists such as bradykinin and acetylcholine in endothelial cells and SMCs.¹⁰³ In endothelial cells, eNOS is predominantly membrane bound,¹⁴⁵ whereas recombinant nNOS is cytosolic¹⁰³ (D. McDonald and K. Channon, unpublished observations, 1999), and it remains to be established whether nNOS in endothelial cells is regulated by mechanisms such as subcellular trafficking, interactions with caveolin and other proteins such as heat shock protein 90,^{88 146} and shear stress–induced activation by Akt-dependent phosphorylation,^{7 8} as described for eNOS. Differential regulation of eNOS and nNOS in target cells in the vascular wall may provide a rationale for choosing one isoform over another for a particular application.

Future work needs to identify the relative advantages of different NOS isoforms for gene therapy in specific vascular diseases or in different target cell types; it is also possible that mutant, chimeric, or otherwise-modified NOS isoforms¹⁴⁷ could offer novel approaches to increasing NO production under conditions of NOS dysregulation.

	Adjunctive Strategies in NOS Gene Transfer

Top Abstract NO and Atherogenesis Biological Effects of NOS... eNOS, nNOS, or iNOS? Adjunctive Strategies in NOS... Summary References

 
The complex mechanisms underlying reduced NO bioactivity in atherosclerosis suggest that the challenge of restoring the vascular NO system lies beyond simply increasing NOS protein in the vessel wall. Intervening in the mechanisms that limit NO production or bioactivity may be alternatives or adjuncts to NOS gene transfer.

Tetrahydrobiopterin is an important modulator of vascular NOS activity and superoxide production.¹⁴⁸ In vitro, the NOS enzyme is strictly dependent on tetrahydrobiopterin for activity and produces superoxide rather than NO when tetrahydrobiopterin levels are inadequate.⁹² The activity of recombinant NOS after gene transfer may be limited by the availability of tetrahydrobiopterin in cell culture,¹⁰³ and some data suggest that tetrahydrobiopterin levels may be limiting in vascular disease in vivo.⁹³ However, NOS gene transfer elevates vascular NO production in vivo without tetrahydrobiopterin supplementation, suggesting that tetrahydrobiopterin is not a limiting factor in target cells in the vessel wall. Nevertheless, increasing vascular tetrahydrobiopterin availability, eg, by gene transfer of GTP–cyclohydrolase I,¹⁴⁹ may represent a powerful complementary strategy to NOS gene therapy in vascular diseases.

The SOD enzymes are a rational approach to increase NO levels. By scavenging superoxide, SOD activity is necessary for NO release from endothelial cells and subsequent interaction with molecular targets.^{79 150} The SODs not only remove superoxide but by generating hydrogen peroxide (H₂O₂) may also contribute to NO actions, eg, by activating guanylate cyclase in VSMCs.¹⁵¹ Adenoviral gene transfer of any of the 3 SOD enzymes (manganese SOD [Mn SOD], copper-zinc SOD [CuZn SOD], or extracellular SOD [EC SOD]) reduces superoxide release from endothelial cells or VSMCs^{152 153 154} and can reduce superoxide-induced LDL oxidation by endothelial cells.¹⁵² In the diabetic rabbit aorta, preliminary data suggested that Mn SOD gene transfer is as effective as eNOS gene transfer in restoring NO-mediated vasorelaxations,¹⁵⁴ but in a more detailed study, neither CuZn SOD nor EC SOD gene transfer to hypercholesterolemic rabbit aortas was sufficient to augment NO-mediated vasorelaxations, despite a reduction in vascular superoxide release.¹⁵³ Similarly, ex vivo SOD gene transfer to diabetic rabbit carotid arteries¹²⁸ or angiotensin II–infused rabbit aortas,¹²⁷ in which vascular superoxide production is increased, did not enhance NO-mediated vasorelaxation, whereas eNOS gene transfer was effective. However, SOD gene transfer and superoxide reduction were most marked at the endothelium, so that SOD gene transfer to deeper layers of the media may be inadequate to affect NO bioactivity.^{127 128 153} Nevertheless, gene transfer of SOD in combination with NOS may provide an approach to simultaneously reduce superoxide release, limit peroxynitrite formation, and potentiate NO bioactivity in vascular disease states.

Gene transfer of NO’s molecular targets is an alternative strategy to increase NO effects. Guanylate cyclase is the principal NO target in VSMCs, leading to cGMP generation and activation of PKG. Adenovirus-mediated gene transfer of both subunits of guanylate cyclase increases cGMP production in VSMCs and reduces the proliferative response to balloon injury.¹⁵⁵ Similarly, PKG gene transfer increases the sensitivity of VSMCs to the antiproliferative and proapoptotic effects of NO,¹⁵⁶ suggesting that these targets may be synergistic with NOS gene transfer in increasing NO-mediated effects.

	Summary

Top Abstract NO and Atherogenesis Biological Effects of NOS... eNOS, nNOS, or iNOS? Adjunctive Strategies in NOS... Summary References

 
Gene therapy for vascular disease remains a future possibility rather than a clinical reality. Nevertheless, the data reviewed here suggest that genetic modification of the vessel wall to increase NOS activity represents a potential approach to clinical problems such as restenosis after balloon angioplasty, venous and arterial bypass grafts, and possibly to high-risk preatherosclerotic conditions or hypertension. Despite encouraging progress in providing proof of concept, however, many important questions remain to be answered to clarify the potential utility of NOS gene therapy in vascular disease. These include identifying the mechanisms of NOS dysregulation in atherosclerosis and assessing the relative importance of superoxide-NO interactions, tetrahydrobiopterin availability, and NO targets in modulating NO bioactivity. Finally, improved gene transfer vectors need to provide longer-term gene expression in vivo, without the confounding and toxic effects of vector-induced inflammation.

	Acknowledgments

 
K.M.C. is funded by the British Heart Foundation (grants FS 93/043 and PG 98/120). S.E.G. is an Established Investigator of the American Heart Association.

Received October 18, 1999; accepted March 28, 2000.

	References

Top Abstract NO and Atherogenesis Biological Effects of NOS... eNOS, nNOS, or iNOS? Adjunctive Strategies in NOS... Summary References

 
1. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta. 1999;1411:217–230.[Medline] [Order article via Infotrieve]

2. Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest. 1997;100:2146–2152.[Medline] [Order article via Infotrieve]

3. Griffith OW, Stuehr DJ. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol. 1995;57:707–736.[Medline] [Order article via Infotrieve]

4. Kibbe M, Billiar T, Tzeng E. Inducible nitric oxide synthase and vascular injury. Cardiovasc Res. 1999;43:650–657.[Abstract/Free Full Text]

5. Prabhakar P, Thatte HS, Goetz RM, Cho MR, Golan DE, Michel T. Receptor-regulated translocation of endothelial nitric-oxide synthase. J Biol Chem. 1998;273:27383–27388.[Abstract/Free Full Text]

6. Michel JB, Feron O, Sacks D, Michel T. Reciprocal regulation of endothelial nitric-oxide synthase by Ca²⁺-calmodulin and caveolin. J Biol Chem. 1997;272:15583–15586.[Abstract/Free Full Text]

7. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399:597–601.[Medline] [Order article via Infotrieve]

8. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601–605.[Medline] [Order article via Infotrieve]

9. Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells can transduce an atheroprotective force. Arterioscler Thromb Vasc Biol. 1998;18:677–685.[Abstract/Free Full Text]

10. Yan Z, Hansson GK. Overexpression of inducible nitric oxide synthase by neointimal smooth muscle cells. Circ Res. 1998;82:21–29.[Abstract/Free Full Text]

11. Wilcox JN, Subramanian RR, Sundell CL, Tracey WR, Pollock JS, Harrison DG, Marsden PA. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 1997;17:2479–2488.[Abstract/Free Full Text]

12. Arthur JF, Yin ZL, Young HM, Dusting GJ. Induction of nitric oxide synthase in the neointima induced by a periarterial collar in rabbits. Arterioscler Thromb Vasc Biol. 1997;17:737–740.[Abstract/Free Full Text]

13. Schwarz PM, Kleinert H, Forstermann U. Potential functional significance of brain-type and muscle-type nitric oxide synthase I expressed in adventitia and media of rat aorta. Arterioscler Thromb Vasc Biol. 1999;19:2584–2590.[Abstract/Free Full Text]

14. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524–526.[Medline] [Order article via Infotrieve]

15. Stamler JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell. 1994;78:931–936.[Medline] [Order article via Infotrieve]

16. Keaney JF Jr, Simon DI, Stamler JS, Jaraki O, Scharfstein J, Vita JA, Loscalzo J. NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties. J Clin Invest. 1993;91:1582–1589.

17. Jia L, Bonaventura C, Bonaventura J, Stamler JS. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature. 1996;380:221–226.[Medline] [Order article via Infotrieve]

18. Gow AJ, Luchsinger BP, Pawloski JR, Singel DJ, Stamler JS. The oxyhemoglobin reaction of nitric oxide. Proc Natl Acad Sci U S A. 1999;96:9027–9032.[Abstract/Free Full Text]

19. Umans JG, Levi R. Nitric oxide in the regulation of blood flow and arterial pressure. Annu Rev Physiol. 1995;57:771–790.[Medline] [Order article via Infotrieve]

20. Lloyd-Jones DM, Bloch KD. The vascular biology of nitric oxide and its role in atherogenesis. Annu Rev Med. 1996;47:365–375.[Medline] [Order article via Infotrieve]

21. Harrison DG. Alterations of vasomotor regulation in atherosclerosis. Cardiovasc Drug Ther. 1995;9(suppl 1):55–63.

22. Radomski MW, Palmer RMJ, Moncada S. The role of nitric oxide and cGMP in platelet adhesion molecules to vascular endothelium. Biochem Biophys Res Commun. 1987;148:1482–1489.[Medline] [Order article via Infotrieve]

23. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774–1777.

24. Groves PH, Banning AP, Penny WJ, Newby AC, Cheadle HA, Lewis MJ. The effects of exogenous nitric oxide on smooth muscle cell proliferation following porcine carotid angioplasty. Cardiovasc Res. 1995;30:87–96.[Medline] [Order article via Infotrieve]

25. Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circ Res. 1996;78:225–230.[Abstract/Free Full Text]

26. Kolpakov V, Gordon D, Kulik TJ. Nitric oxide–generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res. 1995;76:305–309.[Abstract/Free Full Text]

27. Navab M, Hama SY, Nguyen TB, Fogelman AM. Monocyte adhesion molecules and transmigration in atherosclerosis. Coron Artery Dis. 1994;5:198–204.[Medline] [Order article via Infotrieve]

28. Peng H-B, Libby P, Liao JK. Induction and stabilization of IB by nitric oxide mediates inhibition of NF-B. J Biol Chem. 1995;270:14214–14219.[Abstract/Free Full Text]

29. De Caterina R, Libby P, Peng H-B, Thannickal VJ, Rajavashisth TB, Gimbrone MA Jr, Shin WS, Liao JK. Nitric oxide decreases cytokine-induced endothelial activation: nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest. 1995;96:60–68.

30. Gauthier TW, Scalia R, Murohara T, Guo JP, Lefer AM. Nitric oxide protects against leukocyte-endothelium interactions in the early stages of hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1995;15:1652–1659.[Abstract/Free Full Text]

31. Zeiher AM, Fisslthaler B, Schray-Utz B, Busse R. Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells. Circ Res. 1995;76:980–986.[Abstract/Free Full Text]

32. Tsao PS, Wang B, Buitrago R, Shyy JY, Cooke JP. Nitric oxide regulates monocyte chemotactic protein-1. Circulation. 1997;96:934–940.[Abstract/Free Full Text]

33. Heinecke JW. Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis. Curr Opin Lipidol. 1997;8:268–274.[Medline] [Order article via Infotrieve]

34. Hogg N, Kalyanaraman B, Joseph J, Struck A, Parthasarathy S. Inhibition of low-density lipoprotein oxidation by nitric oxide: potential role in atherogenesis. FEBS Lett. 1993;334:170–174.[Medline] [Order article via Infotrieve]

35. Verbeuren TJ, Jordaens FH, Zonnekeyn LL, Van Hove CE, Coene MC, Herman AG. Effect of hypercholesterolemia on vascular reactivity in the rabbit, I: endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits. Circ Res. 1986;58:552–564.[Abstract/Free Full Text]

36. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168–1172.

37. Shimokawa H, Vanhoutte PM. Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis. Circ Res. 1989;64:900–914.[Abstract/Free Full Text]

38. Naruse K, Shimizu K, Muramatsu M, Toki Y, Miyazaki Y, Okumura K, Hashimoto H, Ito T. Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta: PGH₂ does not contribute to impaired endothelium-dependent relaxation. Arterioscler Thromb. 1994;14:746–752.[Abstract/Free Full Text]

39. Cayatte AJ, Palacino JJ, Horten K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14:753–759.[Abstract/Free Full Text]

40. Candipan RC, Wang BY, Buitrago R, Tsao PS, Cooke JP. Regression or progression: dependency on vascular nitric oxide. Arterioscler Thromb Vasc Biol. 1996;16:44–50.[Abstract/Free Full Text]

41. Guo J-P, Milhoan KA, Tuan RS, Lefer AM. Beneficial effect of SPM-5185, a cysteine-containing nitric oxide donor, in rat carotid artery intimal injury. Circ Res. 1994;75:77–84.[Abstract/Free Full Text]

42. Marks DS, Vita JA, Folts JD, Keaney JF, Welch GN, Loscalzo J. Inhibition of neointimal proliferation in rabbits after vascular injury by a single treatment with a protein adduct of nitric oxide. J Clin Invest. 1995;96:2630–2638.

43. Huang PL, Huang ZH, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature. 1995;377:239–242.[Medline] [Order article via Infotrieve]

44. Rudic RD, Shesely EG, Maeda N, Smithies O, Segal SS, Sessa WC. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J Clin Invest. 1998;101:731–736.[Medline] [Order article via Infotrieve]

45. Lake-Bruse KD, Faraci FM, Shesely EG, Maeda N, Sigmund CD, Heistad DD. Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice. Am J Physiol. 1999;277:H770–H776.

46. Mashimo H, Goyal RK. Lessons from genetically engineered animal models, IV: nitric oxide synthase gene knockout mice. Am J Physiol. 1999;277:G745–G750.[Abstract/Free Full Text]

47. Oemar BS, Tschudi MR, Godoy N, Brovkovich V, Malinski T, Luscher TF. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation. 1998;97:2494–2498.[Abstract/Free Full Text]

48. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046–1051.[Abstract]

49. Zeiher AM, Drexler H, Saurbier B, Just H. Endothelium-mediated coronary blood flow modulation in humans: effects of age, atherosclerosis, hypercholesterolemia, and hypertension. J Clin Invest. 1993;92:652–662.

50. Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman SM, Loscalzo J, Dzau VJ. Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J Clin Invest. 1990;86:228–234.

51. Williams SB, Cusco JA, Roddy MA, Johnstone MT, Creager MA. Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Coll Cardiol. 1996;27:567–574.[Abstract]

52. Calver A, Collier J, Moncada S, Vallance P. Effect of local intra-arterial N^G-monomethyl-L-arginine in patients with hypertension: the nitric oxide dilator mechanism appears abnormal. J Hypertens. 1992;10:1025–1031.[Medline] [Order article via Infotrieve]

53. Zeiher AM, Schachinger V, Minners J. Long-term cigarette smoking impairs endothelium-dependent coronary arterial vasodilator function. Circulation. 1995;92:1094–1100.[Abstract/Free Full Text]

54. Clarkson P, Adams MR, Powe AJ, Donald AE, McCredie R, Robinson J, McCarthy SN, Keech A, Celermajer DS, Deanfield JE. Oral L-arginine improves endothelium-dependent dilation in hypercholesterolemic young adults. J Clin Invest. 1996;97:1989–1994.[Medline] [Order article via Infotrieve]

55. Egashira K, Hirooka Y, Kai H, Sugimachi M, Suzuki S, Inou T, Takeshita A. Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia. Circulation. 1994;89:2519–2524.[Abstract/Free Full Text]

56. Drexler H, Zeiher AM, Meinzer K, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolaemic patients by L-arginine. Lancet. 1991;338:1546–1550.[Medline] [Order article via Infotrieve]

57. Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau VJ, Cooke JP. L-arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest. 1992;90:1248–1253.

58. Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996;93:1107–1113.[Abstract/Free Full Text]

59. Buttery LDK, Chester AH, Springall DR, Borland JAA, Michel T, Yacoub MH, Polar JM. Explanted vein grafts with an intact endothelium demonstrate reduced focal expression of endothelial nitric oxide synthase specific to atherosclerotic sites. J Pathol. 1996;179:197–203.[Medline] [Order article via Infotrieve]

60. Liao JK, Shin WS, Lee WY, Clark SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J Biol Chem. 1995;270:319–324.[Abstract/Free Full Text]

61. Bauersachs J, Bouloumi A, Fraccarollo D, Hu K, Busse R, Ertl G. Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation. 1999;100:292–298.[Abstract/Free Full Text]

62. Kerr S, Brosnan MJ, McIntyre M, Reid JL, Dominiczak AF, Hamilton CA. Superoxide anion production is increased in a model of genetic hypertension: role of the endothelium. Hypertension. 1999;33:1353–1358.[Abstract/Free Full Text]

63. Kojda G, Harrison D. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res. 1999;43:562–571.[Medline] [Order article via Infotrieve]

64. Harrison DG. Cellular and molecular mechanisms of endothelial dysfunction. J Clin Invest. 1998;100:2153–2157.[Medline] [Order article via Infotrieve]

65. Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986;320:454–456.[Medline] [Order article via Infotrieve]

66. White CR, Brock TA, Chang L-Y, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MM. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A. 1994;91:1044–1048.[Abstract/Free Full Text]

67. Lefer DJ, Scalia R, Campbell B, Nossuli T, Hayward R, Salamon M, Grayson J, Lefer AM. Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats. J Clin Invest. 1997;99:684–691.[Medline] [Order article via Infotrieve]

68. Darley-Usmar VM, Hogg N, O’Leary VJ, Wilson MT, Moncada S. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Radic Res Commun. 1992;17:9–20.[Medline] [Order article via Infotrieve]

69. Ushio-Fukai M, Alexander RW, Akers M, Yin QQ, Fujio Y, Walsh K, Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem. 1999;274:22699–22704.[Abstract/Free Full Text]

70. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD. Cell transformation by the superoxide-generating oxidase Mox1. Nature. 1999;401:79–82.[Medline] [Order article via Infotrieve]

71. Mugge A, Brandes RP, Boger RH, Dwenger A, Bode-Boger S, Kienke S, Frolich JC, Lichtlen PR. Vascular release of superoxide radicals is enhanced in hypercholesterolemic rabbits. J Cardiovasc Pharmacol. 1994;24:994–998.[Medline] [Order article via Infotrieve]

72. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993;91:2546–2551.

73. Warnholtz A, Nickenig G, Schulz E, Macharzina R, Brasen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Bohm M, Meinertz T, Muenzel T. Increased NADH-oxidase–mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation. 1999;99:2027–2033.[Abstract/Free Full Text]

74. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NAD/NADPH oxidase activation. J Clin Invest. 1996;97:1916–1923.[Medline] [Order article via Infotrieve]

75. Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers QT, Taylor WR, Harrison DG, de Leon H, Wilcox JN, Griendling KK. p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res. 1997;80:45–51.[Abstract/Free Full Text]

76. Ohara Y, Peterson TE, Sayegh HS, Subramanian RR, Wilcox JN, Harrison DG. Dietary correction of hypercholesterolemia in the rabbit normalizes endothelial superoxide anion production. Circulation. 1995;92:898–903.[Abstract/Free Full Text]

77. Keaney JF, Vita JA. Atherosclerosis, oxidative stress, and antioxidant protection in endothelium-derived relaxing factor action. Prog Cardiovasc Dis. 1995;38:129–154.[Medline] [Order article via Infotrieve]

78. Mugge A, Elwell JH, Peterson TE, Hofmeyer TG, Heistad DD, Harrison DG. Chronic treatment with polyethylene-glycolated superoxide dismutase partially restores endothelium-dependent vascular relaxations in cholesterol-fed rabbits. Circ Res. 1991;69:1293–1300.[Abstract/Free Full Text]

79. Mugge A, Elwell JH, Peterson TE, Harrison DG. Release of intact endothelium-derived relaxing factor depends on endothelial superoxide dismutase activity. Am J Physiol. 1991;260:C219–C225.[Abstract/Free Full Text]

80. Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. 1995;77:510–518.[Abstract/Free Full Text]

81. Cosentino F, Hishikawa K, Katusic ZS, Luscher TF. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation. 1997;96:25–28.[Abstract/Free Full Text]

82. Xia Y, Dawson VL, Dawson TM, Snyder SH, Zweier JL. Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc Natl Acad Sci U S A. 1996;93:6770–6774.[Abstract/Free Full Text]

83. Pou S, Keaton L, Surichamorn W, Rosen GM. Mechanism of superoxide generation by neuronal nitric oxide synthase. J Biol Chem. 1999;274:9573–9580.[Abstract/Free Full Text]

84. Rizzo V, McIntosh DP, Oh P, Schnitzer JE. In situ flow activates endothelial nitric oxide synthase in luminal caveolae of endothelium with rapid caveolin dissociation and calmodulin association. J Biol Chem. 1998;273:34724–34729.[Abstract/Free Full Text]

85. Peterson TE, Poppa V, Ueba H, Wu A, Yan C, Berk BC. Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae. Circ Res. 1999;85:29–37.[Abstract/Free Full Text]

86. Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest. 1999;103:897–905.[Medline] [Order article via Infotrieve]

87. Blair A, Shaul PW, Yuhanna IS, Conrad PA, Smart EJ. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation. J Biol Chem. 1999;274:32512–32519.[Abstract/Free Full Text]

88. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature. 1998;392:821–824.[Medline] [Order article via Infotrieve]

89. Fleming I, Busse R. Signal transduction of eNOS activation. Cardiovasc Res. 1999;43:532–541.[Abstract/Free Full Text]

90. Schmidt K, Werner ER, Mayer B, Wachter H, Kukovetz WR. Tetrahydrobiopterin-dependent formation of endothelium-derived relaxing factor (nitric oxide) in aortic endothelial cells. Biochem J. 1992;281:297–300.

91. Wever RMF, van Dam T, van Rijn HJ, de Groot F, Rabelink TJ. Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem Biophys Res Commun. 1997;237:340–344.[Medline] [Order article via Infotrieve]

92. Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, Pritchard KA Jr. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A. 1998;95:9220–9225.[Abstract/Free Full Text]

93. Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, Masada M, Toda N, Kikkawa R. Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O₂-imbalance in insulin-resistant rat aorta. Diabetes. 1999;48:2437–2445.[Abstract]

94. Cosentino F, Patton S, d’Uscio LV, Werner ER, Werner-Felmayer G, Moreau P, Malinski T, Luscher TF. Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats. J Clin Invest. 1998;101:1530–1537.[Medline] [Order article via Infotrieve]

95. Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, Luscher T, Rabelink T. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest. 1997;99:41–46.[Medline] [Order article via Infotrieve]

96. Heitzer T, Brockhoff C, Mayer B, Warnholtz A, Mollnau H, Henne S, Meinertz T, Munzel T. Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: evidence for a dysfunctional nitric oxide synthase. Circ Res. 2000;86:E36–E41.

97. Kinoshita H, Milstien S, Wambi C, Katusic ZS. Inhibition of tetrahydrobiopterin biosynthesis impairs endothelium-dependent relaxations in canine basilar artery. Am J Physiol. 1997;273:H718–H724.[Abstract/Free Full Text]

98. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, Imaizumi T. Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation. 1999;99:1141–1146.[Abstract/Free Full Text]

99. Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, Blaschke TF, Cooke JP. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation. 1998;98:1842–1847.[Abstract/Free Full Text]

100. Bode-Boger SM, Boger RH, Kienke S, Junker W, Frolich JC. Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits. Biochem Biophys Res Commun. 1996;219:598–603.[Medline] [Order article via Infotrieve]

101. Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res. 1999;43:542–548.[Abstract/Free Full Text]

102. Kullo IJ, Simari RD, Schwartz RS. Vascular gene transfer: from bench to bedside. Arterioscler Thromb Vasc Biol. 1999;19:196–207.[Free Full Text]

103. Channon KM, Blazing MA, Shetty GA, Potts KE, George SE. Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells. Cardiovasc Res. 1996;32:962–972.[Medline] [Order article via Infotrieve]

104. Ooboshi H, Chu Y, Rios CD, Faraci FM, Davidson BL, Heistad DD. Altered vascular function after adenovirus-mediated overexpression of endothelial nitric oxide synthase. Am J Physiol. 1997;273:H265–H270.[Abstract/Free Full Text]

105. Ooboshi H, Toyoda K, Faraci FM, Lang MG, Heistad DD. Improvement of relaxation in an atherosclerotic artery by gene transfer of endothelial nitric oxide synthase. Arterioscler Thromb Vasc Biol. 1998;18:1752–1758.[Abstract/Free Full Text]

106. Mozes G, Kullo IJ, Mohacsi TG, Cable DG, Spector DJ, Crotty TB, Gloviczki P, Katusic ZS, O’Brien T. Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine. Atherosclerosis. 1998;141:265–271.[Medline] [Order article via Infotrieve]

107. Cable DG, O’Brien T, Kullo IJ, Schwartz RS, Schaff HV, Pompili VJ. Expression and function of a recombinant endothelial nitric oxide synthase gene in porcine coronary arteries. Cardiovasc Res. 1997;35:553–559.[Abstract/Free Full Text]

108. Chen AF, O’Brien T, Tsutsui M, Kinoshita H, Pompili VJ, Crotty TB, Spector DJ, Katusic ZS. Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery. Circ Res. 1997;80:327–335.[Abstract/Free Full Text]

109. Cable DG, Caccitolo JA, Pearson PJ, O’Brien T, Mullany CJ, Daly RC, Orszulak TA, Schaff HV. New approaches to prevention and treatment of radial artery graft vasospasm. Circulation. 1998;98(suppl II):II-15–II-21.

110. Cable DG, O’Brien T, Schaff HV, Pompili VJ. Recombinant endothelial nitric oxide synthase transduced human saphenous veins: gene therapy to augment nitric oxide production in bypass conduits. Circulation. 1997;96(suppl II):II-173–II-178.

111. Tsutsui M, Chen AF, O’Brien T, Crotty TB, Katusic ZS. Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium. Arterioscler Thromb Vasc Biol. 1998;18:1231–1241.[Abstract/Free Full Text]

112. Janssens S, Flaherty D, Nong Z, Varenne O, van Pelt N, Haustermans C, Zoldhelyi P, Gerard R, Collen D. Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats. Circulation. 1998;97:1274–1281.[Abstract/Free Full Text]

113. Von der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci U S A. 1995;92:1137–1141.[Abstract/Free Full Text]

114. Fang S, Sharma RV, Bhalla RC. Enhanced recovery of injury-caused downregulation of paxillin protein by eNOS gene expression in rat carotid artery: mechanism of NO inhibition of intimal hyperplasia? Arterioscler Thromb Vasc Biol. 1999;19:147–152.[Abstract/Free Full Text]

115. Shears LL, Kibbe MR, Murdock AD, Billiar TR, Lizonova A, Kovesdi I, Watkins SC, Tzeng E. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J Am Coll Surg. 1998;187:295–306.[Medline] [Order article via Infotrieve]

116. Varenne O, Pislaru S, Gillijns H, Van Pelt N, Gerard RD, Zoldhelyi P, Van de Werf F, Collen D, Janssens SP. Local adenovirus-mediated transfer of human endothelial nitric oxide synthase reduces luminal narrowing after coronary angioplasty in pigs. Circulation. 1998;98:919–926.[Abstract/Free Full Text]

117. Channon KM, Qian HS, Neplioueva V, Blazing MA, Olmez E, Shetty GA, Youngblood SA, Stamler JS, George SE. In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-fed rabbits. Circulation. 1998;98:1905–1911.[Abstract/Free Full Text]

118. Qian HS, Neplioueva V, Shetty GA, Channon KM, George SE. Nitric oxide synthase gene therapy rapidly reduces adhesion molecule expression and inflammatory cell infiltration in carotid arteries of cholesterol-fed rabbits. Circulation. 1999;99:2979–2982.[Abstract/Free Full Text]

119. Kullo IJ, Mozes G, Schwartz RS, Gloviczki P, Tsutsui M, Katusic ZS, O’Brien T. Enhanced endothelium-dependent relaxations after gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries. Hypertension. 1997;30:314–320.[Abstract/Free Full Text]

120. Kullo IJ, Mozes G, Schwartz RS, Gloviczki P, Crotty TB, Barber DA, Katusic ZS, O’Brien T. Adventitial gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity. Circulation. 1997;96:2254–2261.[Abstract/Free Full Text]

121. Chen AFY, Jiang SW, Crotty TB, Tsutsui M, Smith LA, O’Brien T, Katusic ZS. Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries. Proc Natl Acad Sci U S A. 1997;94:12568–12573.[Abstract/Free Full Text]

122. Shears LL, Kawaharada N, Tzeng E, Billiar TR, Watkins SC, Kovesdi I, Lizonova A, Pham SM. Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis. J Clin Invest. 1997;100:2035–2042.[Medline] [Order article via Infotrieve]

123. Matsumoto T, Komori K, Yonemitsu Y, Morishita R, Sueishi K, Kaneda Y, Sugimachi K. Hemagglutinating virus of Japan-liposome-mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff. J Vasc Surg. 1998;27:135–144.[Medline] [Order article via Infotrieve]

124. Janssens SP, Bloch KD, Nong Z, Gerard RD, Zoldhelyi P, Collen D. Adenoviral-mediated transfer of the human endothelial nitric oxide synthase gene reduces acute hypoxic pulmonary vasoconstriction in rats. J Clin Invest. 1996;98:317–324.[Medline] [Order article via Infotrieve]

125. Gaballa MA, Goldman S. Overexpression of endothelium nitric oxide synthase reverses the diminished vasorelaxation in the hindlimb vasculature in ischemic heart failure in vivo. J Mol Cell Cardiol. 1999;31:1243–1252.[Medline] [Order article via Infotrieve]

126. Alexander MY, Brosnan MJ, Hamilton CA, Downie P, Devlin AM, Dowell F, Martin W, Prentice HM, O’Brien T, Dominiczak AF. Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model. Cardiovasc Res. 1999;43:798–807.[Abstract/Free Full Text]

127. Nakane H, Miller FJ Jr, Faraci FM, Toyoda K, Heistad DD. Gene transfer of endothelial nitric oxide synthase reduces angiotensin II–induced endothelial dysfunction. Hypertension. 2000;35:595–601.[Abstract/Free Full Text]

128. Lund DD, Faraci FM, Miller FJ Jr, Heistad DD. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation. 2000;101:1027–1033.[Abstract/Free Full Text]

129. Champion HC, Bivalacqua TJ, D’Souza FM, Ortiz LA, Jeter JR, Toyoda K, Heistad DD, Hyman AL, Kadowitz PJ. Gene transfer of endothelial nitric oxide synthase to the lung of the mouse In vivo: effect on agonist-induced and flow-mediated vascular responses. Circ Res. 1999;84:1422–1432.[Abstract/Free Full Text]

130. Myers PR, Tanner MA. Vascular endothelial cell regulation of extracellular matrix collagen: role of nitric oxide. Arterioscler Thromb Vasc Biol. 1998;18:717–722.[Abstract/Free Full Text]

131. Tarry WC, Makhoul RG. L-Arginine improves endothelium-dependent vasorelaxation and reduces intimal hyperplasia after balloon angioplasty. Arterioscler Thromb. 1994;14:938–943.[Abstract/Free Full Text]

132. West NEJ, Qian HS, Citrin D, George SE, Channon KM. Intraoperative adenoviral gene transfer of nitric oxide synthase reduces early inflammation and intimal hyperplasia in venous bypass grafts. Heart. 1999;81(suppl 1):17. Abstract.

133. Guo K, Andres V, Walsh K. Nitric oxide–induced downregulation of Cdk2 activity and cyclin A gene transcription in vascular smooth muscle cells. Circulation. 1998;97:2066–2072.[Abstract/Free Full Text]

134. Sharma RV, Tan E, Fang S, Gurjar MV, Bhalla RC. NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells. Am J Physiol. 1999;276:H1450–H1459.[Abstract/Free Full Text]

135. Gurjar MV, Sharma RV, Bhalla RC. eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity. Arterioscler Thromb Vasc Biol. 1999;19:2871–2877.[Abstract/Free Full Text]

136. Pollman MJ, Yamada T, Horiuchi M, Gibbons GH. Vasoactive substances regulate vascular smooth muscle cell apoptosis: countervailing influences of nitric oxide and angiotensin II. Circ Res. 1996;79:748–756.[Abstract/Free Full Text]

137. Meng YY, Trachtenburg J, Ryan US, Abendschein DR. Potentiation of endogenous nitric oxide with superoxide dismutase inhibits platelet-mediated thrombosis in injured and stenotic arteries. J Am Coll Cardiol. 1995;25:269–275.[Abstract]

138. Niebauer J, Dulak J, Chan JR, Tsao PS, Cooke JP. Gene transfer of nitric oxide synthase: effects on endothelial biology. J Am Coll Cardiol. 1999;34:1201–1207.[Abstract/Free Full Text]

139. Tsutsui M, Onoue H, Iida Y, Smith L, O’Brien T, Katusic ZS. Adventitia-dependent relaxations of canine basilar arteries transduced with recombinant eNOS gene. Am J Physiol. 1999;276:H1846–H1852.

140. Ooboshi H, Welsh MJ, Rios CD, Davidson BL, Heistad DD. Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue. Circ Res. 1995;77:7–13.[Abstract/Free Full Text]

141. Christenson SD, Lake KD, Ooboshi H, Faraci FM, Davidson BL, Heistad DD. Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue in mice. Stroke. 1998;29:1411–1415.[Abstract/Free Full Text]

142. Hansson GK, Geng Y, Holm J, Hårdhammar P, Wennmalm, Jennische E. Arterial smooth muscle cells express nitric oxide synthase in response to endothelial injury. J Exp Med. 1994;180:733–738.[Abstract/Free Full Text]

143. Banning AP, Groves PH, Buttery LD, Wharton J, Rutherford RA, Black P, Winkler F, Polak JM, Lewis MJ, Drexler H. Reciprocal changes in endothelial and inducible nitric oxide synthase expression following carotid angioplasty in the pig. Atherosclerosis. 1999;145:17–32.[Medline] [Order article via Infotrieve]

144. Xia Y, Roman LJ, Masters BS, Zweier JL. Inducible nitric-oxide synthase generates superoxide from the reductase domain. J Biol Chem. 1998;273:22635–22639.[Abstract/Free Full Text]

145. Michel T, Li GK, Busconi L. Phosphorylation and subcellular translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1993;90:6252–6256.[Abstract/Free Full Text]

146. Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA, Michel T. Endothelial nitric oxide synthase targeting to caveolae: specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells. J Biol Chem. 1996;271:22810–22814.[Abstract/Free Full Text]

147. Nishida CR, Ortiz de Montellano PR. Electron transfer and catalytic activity of nitric oxide synthases: chimeric constructs of the neuronal, inducible, and endothelial isoforms. J Biol Chem. 1998;273:5566–5571.[Abstract/Free Full Text]

148. Cosentino F, Luscher TF. Tetrahydrobiopterin and endothelial function. Eur Heart J. 1998;19:G3–G8.

149. Kibbe MR, Nie S, Yoneyama T, Hatakeyama K, Lizonova A, Kovesdi I, Billiar TR, Tzeng E. Optimization of ex vivo inducible nitric oxide synthase gene transfer to vein grafts. Surgery. 1999;126:323–329.[Medline] [Order article via Infotrieve]

150. Omar HA, Cherry PD, Mortelliti MP, Burke-Wolin T, Wolin MS. Inhibition of coronary artery superoxide dismutase attenuates endothelium-dependent and -independent nitrovasodilator relaxation. Circ Res. 1991;69:601–608.[Abstract/Free Full Text]

151. Mohazzab HK, Kaminski PM, Fayngersh RP, Wolin MS. Oxygen-elicited responses in calf coronary arteries: role of H₂O₂ production via NADH-derived superoxide. Am J Physiol. 1996;270:H1044–H1053.[Abstract/Free Full Text]

152. Fang X, Weintraub NL, Rios CD, Chappell DA, Zwacka RM, Engelhardt JF, Oberley LW, Yan T, Heistad DD, Spector AA. Overexpression of human superoxide dismutase inhibits oxidation of low-density lipoprotein by endothelial cells. Circ Res. 1998;82:1289–1297.[Abstract/Free Full Text]

153. Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998;82:1298–1305.[Abstract/Free Full Text]

154. Zanetti M, Sato J, Katusic ZS, O’Brien T. Gene transfer of endothelial nitric oxide synthase and superoxide dismutase improves vascular reactivity of aortas from diabetic rabbits. Circulation. 1999;100(suppl I):I-624. Abstract.

155. Sinnaeve P, Chiche JD, Nong Z, Varenne O, Van Pelt N, Gerard RD, Collen D, Bloch K, Janssens S. Adenovirus-mediated transfer of soluble guanylate cyclase 1 and ß1 subunits increases nitric oxide responsiveness and reduces neointima formation in balloon-injured rat carotid arteries. Circulation. 1998;98(suppl I):I-319. Abstract.

156. Chiche JD, Schlutsmeyer SM, Bloch DB, de la Monte SM, Roberts JD Jr, Filippov G, Janssens SP, Rosenzweig A, Bloch KD. Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem. 1998;273:34263–34271.[Abstract/Free Full Text]

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