Arch Pharm Res Vol 32, No 8, 1103-1108, 2009DOI 10.1007/s12272-009-1801-1

1103

Nitric Oxide and Pathogenic Mechanisms Involved in the Develop-ment of Vascular Diseases

Claudio Napoli1 and Louis J. Ignarro2,3

1Department of General Pathology and Excellence Research Center on Cardiovascular Diseases, Chair of Clinical Pathol-ogy, 1St School of Medicine, II University of Naples, 80138 Naples, Italy, 2Department of Medical and Molecular Phar-macology, University of California Los Angeles, David Geffen School of Medicine, Center for Health Sciences, Los Angeles,California 90095-1735, USA, and 3WCU program, School of Medicine, Konkuk University, Seoul 143-701, Korea

(Received February 25, 2009/Revised March 17, 2009/Accepted June 25, 2009)

Nitric oxide (NO) is a pivotal signaling messenger in the cardiovascular system. NO partici-pates in regulatory functions including control of hemostasis, fibrinolysis, platelet and leuko-cyte interactions with the arterial wall, regulation of vascular tone, proliferation of vascularsmooth muscle cells, and homeostasis of blood pressure. Diminished NO bioavailability andabnormalities in NO-dependent signaling are among central factors of vascular disease,although it is unclear whether this is a cause of, or result of endothelial dysfunction or bothpathogenic events. Disturbances in NO bioavailability have been linked to cause endothelialdysfunction, leading to increased susceptibility to atherosclerotic lesion progression, hyperten-sion, hypercholesterolemia, diabetes mellitus, thrombosis and stroke. Key words: Nitric oxide, Atherosclerosis, Smooth muscle cells

INTRODUCTION

Nitric oxide (NO) acts as a central signal trans-duction pathway in endothelium (Ignarro et al.,1999). Specifically, NO regulates hemostasis offibrinolysis, regulation of vascular tone, prolifera-tion of vascular smooth muscle cells (VSMCs), andblood pressure (Napoli and Ignarro, 2001; Napoli etal., 2006; Rabelink and Luscher, 2006). ReducedNO bioavailability is implicated in the developmentof vascular disease, although it is poorly understoodwhether this is a cause of, or result of endothelialdysfunction or both pathogenic events. Distur-bances in NO pathway can cause endothelial dys-function, leading to increased susceptibility toatherosclerosis, hypertension, hypercholesterolemia,diabetes mellitus, thrombosis and cerebrovasculardisease (Napoli and Ignarro, 2001; Napoli et al.,2006).

NO is produced by a family of NO synthase (NOS)enzymes (Napoli and Ignarro, 2001; Napoli et al.,2006), of which three main isoforms, encoded onseparate chromosomes by separate genes, have beenidentified in human beings and other organisms(Napoli and Ignarro, 2001; Napoli et al., 2006): a)neuronal NOS (nNOS) predominantly expressed incertain neurons and in skeletal muscle; b) endo-thelial NOS (eNOS) predominantly expressed inendothelial cells, and c) inducible NOS (iNOS)expressed by macrophage/monocyte lineage cells.The three NOS isoforms have similar enzymaticmechanisms that involve electron transfer for oxi-dation of the terminal guanidin nitrogen of L-argi-nine. These enzymes all require several cofactorsfor proper function, including tetrahydrobiopterin(BH4), nicotinamide-adenine-dinucleotide phosphate(NADPH), flavin adenine dinucleotide, and flavinmononucleotide. Mice lacking the endothelial isoformare hypertensive, have endothelial dysfunction andshow a more severe outcome in response to vascularinjury, to cerebral ischemia, and to hypercholester-olemic diet-induced atherogenesis (Liu and Huang,2008). Mice lacking the neuronal isoform show a

Correspondence to: Claudio Napoli, Department of GeneralPathology, 1St School of Medicine, Via Costantinopoli 16, II Uni-versity of Naples, 80138 Naples, ItalyE-mail claudio.napoli@unina2.it

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less severe outcome in response to cerebral ischemiabut have increased diet-induced atherosclerosiswhile mice lacking the inducible isoform show re-duced hypotension to septic shock (Liu and Huang,2008).The physiologic target of NO is solubleguanylate cyclase (Napoli and Ignarro, 2001; Napoliet al., 2006). NO activates guanylate cyclase bybinding to its heme moiety, resulting in increasedcGMP levels. In the vasculature, cGMP mediatesNO-dependent relaxation of VSMC, resulting invasodilation. Similarly, NO produced as a neuro-transmitter in the gastrointestinal, urinary, andthe respiratory tract mediates smooth muscle re-laxation by increasing in cGMP production. Theseeffects are likely mediated by the phosphorylationof downstream proteins by cGMP-dependent proteinkinases, including myosin light chain (Napoli andIgnarro, 2001; Napoli et al., 2006). Another targetfor NO is sulfhydryl groups on proteins, to form S-nitrosothiol (SNOs) compounds. Currently, increas-ing evidence indicates that NO mediates a plethoraof actions through cGMC-independent mechanisms(Ignarro et al., 1999; Napoli and Ignarro, 2001; Napoliet al., 2006; Rabelink and Luscher, 2006; Liu andHuang, 2008).

NO IN THE DEVELOPMENT OF VASCU-LAR DISEASE

An early event in the pathophysiology of athero-sclerosis is the impairment of endothelial functionbefore structural changes such as intimal hyper-plasia or lipid deposition occur. Diminished levelsof bioavailable NO, one of the hallmarks of endo-thelial dysfunction, occur through several potentialmechanisms, such as reduced eNOS expression levels,reduced eNOS enzymatic activity, and reduced NObioavailability (Napoli and Ignarro, 2001; Napoli etal., 2006; Liu and Huang, 2008). Endothelial dys-function is associated with an increase in ROS pro-duction in the vasculature. Activation of the endo-thelial cell NADPH oxidase and formation of per-oxynitrite during angiotensin II induced mitochon-drial dysfunction modulates endothelial NO andsuperoxide generation, which in turn has ramifica-tions for development of endothelial dysfunction(Doughan et al., 2008). Thus, coronary heart dise-ase (CHD) risk factors that deplete levels of L-arginine or BH4 may promote NOS-mediated ROSformation, and in turn, increase peroxynitrite gen-eration (Ignarro and Napoli, 2004). Clinically, someof the CHD risk factors identified with endothelialdysfunction are associated with decreased bioavail-

able NO, as evidenced by an abnormal coronaryvasodilator response to acetylcholine challenge (Napoliand Ignarro, 2001; Napoli et al., 2006). Moreoverrecent findings indicate that coronary endothelialdysfunction in humans is characterized by localenhancement of oxidative stress without a decreasein basal NO release and support the hypothesisthat local oxidative stress has a role in reduction ofNO bioavailability in humans with coronary endo-thelial dysfunction (Lavi et al., 2008; Schiffrin, 2008).

NO AND OXIDATION-SENSITIVE MECHA-NISMS

An extensive exploration of the oxidation-depen-dent mechanisms has been widely described (Napoliand Ignarro, 2001; Ignarro and Napoli, 2004; Napoliand Lerman, 2001; de Nigris et al., 2003). Athero-genic lipids, particularly oxidized low density lipo-proteins (oxLDL), are responsible for a wide rangeof cellular dysfunctions within the vessel wall,playing a pivotal role in human early atherogenesis(Napoli and Ignarro, 2001; Ignarro and Napoli, 2004;Napoli and Lerman, 2001; de Nigris et al., 2003).Indeed, oxLDL induces monocyte adhesion to theendothelium, migration and proliferation of smoothmuscle cells, injures cells, interferes with NO release,and promotes procoagulant properties of vascularcells. Native and oxLDL can uncouple eNOS andmay also induce a decreased uptake of L-arginine.The local depletion of the L-arginine substrate mayderange the eNOS, leading to overproduction of super-oxide radical from oxygen, the co-substrate of eNOS(Napoli and Ignarro, 2001; Ignarro and Napoli,2004; Napoli and Lerman, 2001; de Nigris et al.,2003).

Moreover, oxLDL increases the availability andactivity of arginase II (at both transcriptional andposttranslational levels), reciprocally decreases NOxproduction, and contributes to impaired vascularNO signaling. The mechanism for the activation ofarginase within the endothelial cell involves disso-ciation of arginase II from microtubules, a key mecha-nism in early arginase II activation. Interestingly,physiological differences can affect arterial segmentsfrom different regions (Napoli et al., 1997; Napoli etal., 1999; D’Armiento et al., 2001). For example,oxLDL impairs contraction and endothelium-de-pendent relaxation in carotid but not in basilar artery(Napoli et al., 1997), suggesting that intracranialarteries may be relatively protected from athero-sclerosis via endothelial resistance to oxidativeinjury. Finally, a multitude of oxidation-sensitive

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apoptotic signaling can interact with NO in thearterial wall (Napoli and Lerman, 2001; Napoli etal., 2002). Thus, the balance between NO bioacti-vity and oxidative stress plays an important role inthe development of atherogenesis. This conceptdemonstrates that L-arginine hypothesis and theincreased oxidative stress may actually fit togetherand are not two concepts which exclude each other.

A consistent number of studies demonstrate thateNOS gene polymorphism can be considered anadditional risk factor that contributes to endo-thelial dysfunction and atherosclerosis in manycardiovascular events (Napoli et al., 2006). The twopolymorphisms located in the exon 7 (894 G→Twhich encodes a Glu298→Asp amino acid substitu-tion in eNOS gene) and in the promoter region (T-786→C) of the eNOS gene are associated withfunctional changes in the endothelium and carotidintima-media thickness (IMT), mild modulation ofthe predisposition abdominal aortic aneurysm(reviewed in Napoli et al., 2006; Napoli and Ignarro,2007) and could be a risk factor for angiographicCHD and recent myocardial infarction reviewed inNapoli et al., 2006).

MOLECULAR MECHANISMS REGULA-TING eNOS

The molecular mechanisms regulating eNOS in-volve both genomic and non-genomic pathophy-siological mechanisms (Napoli and Ignarro, 2001;Napoli et al., 2006). The eNOS promoter gene pos-sesses consensus sequences that are potentialbinding sites for transcription/nuclear factors suchas AP-1 complex, NF-κB, and IL-6 (de Nigris et al.,2003). Abundance of eNOS is increased by phy-siological shear stress, while it is decreased by lipo-proteins (LDL), angiotensin-II, and tumor necrosisfactor α by a decreased mRNA stability. eNOS is atightly coupled enzyme system that may be easilydysregulated by perturbations in availability ofsubstrates and cofactors as well as by competitiveinhibitors such as ADMA (Napoli and Ignarro,2001; Napoli et al., 2006). Uncoupling of eNOS alsoresults in increased endothelial production of su-peroxide and the conversion of NO to peroxynitrite,a powerful pro-oxidant that reduces eNOS bioacti-vity. Recoupling of eNOS with L-arginine, tetrahy-drobiopterin, and antioxidant supplements are ther-apeutical approaches for augmenting the favourableeffects of peroxisome proliferator-activated receptors(PPARs) on eNOS (Ignarro et al., 2002; Napoli andIgnarro, 2003). Other pharmacotherapies, such as

statins, ACE inhibitors, angiotensin II receptorblockers, and calcium channel blockers can regulateeNOS activity by both genomic and non-genomicmechanisms (Napoli and Ignarro, 2001; Napoli etal., 2006; Ignarro et al., 2002; Napoli and Ignarro,2003).

The suppression of inflammatory signaling path-ways by PPAR-α activation provides an additionalmechanism whereby fenofibrate, a specific PPAR-αagonist, could influence eNOS bioactivity (Goya etal., 2004). Fenofibrate seems to increase the mRNAexpression, protein level, and enzyme activity ofeNOS in a dose-dependent manner. However, theeNOS promoter sequence does not possess a PPARresponse element indicating that fenofibrate did notenhance eNOS promoter activity (Goya et al.,2004). Moreover, in mRNA stability assays, fenofi-brate increases the half-life of eNOS mRNA. Theobservation that PPAR-α agonists stabilize mRNAlevels is unexpected and raises the question ofwhether these effects occur via PPAR-α. Furtherstudies employing gene knock-down technologyand/or in vivo analysis on PPAR-α-deficient miceare required to address this point (Goya et al.,2004). PPAR-α activation can also have direct anti-atherogenic effects on the different cell types of thevascular wall by decreasing the expression of adhe-sion molecules, tissue factor, interleukin-6 (IL-6),and endothelin-1 (Israelian-Konaraki and Reaven,2005). The latest evidence describing how PPARtranscription factors may modulate different stepsof atherosclerosis development and progression andthe therapeutical potential of PPAR ligands havebeen recently reviewed elsewhere (Bouhlel et al.,2008).

Over the last several years, there has been a con-siderable amount of research implicating Krüppel-like transcription factor 2 (KLF2) as key regulatorsof the endothelial proinflammatory pathways (Atkinsand Jain, 2007). KLF2 differentially regulates keyfactors involved in maintaining an antithromboticendothelial surface. Indeed, KLF2 is inhibited bythe inflammatory cytokine interleukin-1β (IL-1β),mediated the beneficial effects of statins in endo-thelial cells, and is induced by laminar shear stressin endothelial cells (Napoli et al., 2006). Overexpres-sion of KLF2 strongly induces eNOS expressionand may inhibit the proinflammatory cytokines-dependent induction of vascular cell adhesion mole-cule-1 (VCAM-1) and endothelial adhesion moleculeE-selectin (Napoli et al., 2006). In another study,KLF2 was found to strongly induce thrombomodulin(TM) and eNOS expression and reduce plasminogen

1106 C. Napoli and L. J. Ignarro

activator inhibitor-1 (PAI-1) expression (Lin et al.,2005).

In situ hybridization approaches revealed thatKLF2 expression is limited to the endothelial layerof the human aorta and, importantly, KLF2 expres-sion is decreased at branch points (Sen-Banerjee etal., 2004). These findings are intriguing, as patholo-gic studies have identified arterial branch points asthe earliest atheroprone regions of the human vas-culature. More recently, KLF2 has been implicatedin the activation of protein 1 (AP-1) pathway (Boonet al., 2007; Fledderus et al., 2007) and in theinhibition of transforming growth factor (TGF)-βsignaling via 2 distinct mechanisms (Boon et al.,2007). Using overexpression and knockdown studies,KLF2 was shown to induce Smad7, subsequentlysuppressing Smad2 phosphorylation and Smad3/4-dependent transcriptional activation. In addition,KLF2 simultaneously inhibits the TGF-β signalingcofactor AP-1 (Boon et al., 2007). Human endothelialcells overlying atherosclerotic plaques exhibit in-creased levels of phosphorylated nuclear activatingtranscription factor 2 (ATF2), which is among theheterodimeric components of AP-1 (Boon et al., 2007).Using knockdown and overexpression studies, shearstress suppressed nuclear levels of activated ATF2by inhibiting its nuclear translocation via KLF2.

Studies by SenBanerjee et al. (Sen-Banerjee etal., 2004) were the first to identify KLF2 as a potentinducer of eNOS expression and activity showingthat eNOS promoter deletion and mutational analy-sis revealed a single KLF2 site to be critical for theability of KLF2 to bind and activate the eNOS pro-moter (Sen-Banerjee et al., 2004). This activationfunction is mediated by KLF2 and its recruitmentof the coactivator CBP/p300 to the eNOS promoter(Sen-Banerjee et al., 2004). Multiple studies haveconfirmed the ability of KLF2 to induce eNOS anddemonstrated that KLF2 can inhibit the expressionimportant genes in regulating vessel tone, such asendothelin, adrenomedullin, and angiotensin-con-verting enzyme (Parmar et al., 2006; Dekker et al.,2005, 2006). Genomic profiling studies identifiedthe ability of KLF2 to induce C-natriuretic peptideand arginosuccinate synthase, a limiting enzyme ineNOS substrate bioavailability (Parmar et al., 2005;Goodwin et al., 2004). Moreover, loss-of-functionstudies using siRNA approaches have offered im-portant validation of the effects of KLF2. Indeed,knockdown of KLF2 leads to reduced expression ofeNOS and C-natriuretic peptide under basal andflow conditions (Parmar et al., 2006; Dekker et al.,2005).

Post-translational covalent modification of eNOSis also fundamentally important in the homeostasisof NO, chiefly by regulating the subcellular locali-zation of the enzyme. The deacylation/reacylationreaction of eNOS in the plasmalemmal caveolaeregulates NO production (Napoli et al., 2006; Limet al., 2007; Hayashi et al., 2007). Acylation targetsthe localization of eNOS to plasmalemmal caveolae,a site where the enzyme activity is inhibited throughassociation with caveolin. Increase in cytosolic [Ca2+]in response to acetylcholine-induced activation ofcell surface receptors, induces the allosteric bindingof calmodulin to eNOS. The enzyme then dissociatesfrom caveolin and starts to generate NO (Napoli etal., 2006). eNOS in caveolae is held inactive by itsassociation with caveolin-1, but eNOS activity canbe increased by Ca2+/calmodulin and binding toHSP90 and dynamin-2 (Cao et al., 2003; Garcia-Cardena et al., 1998). HSP90 facilitates the phos-phorylation of eNOS by forming a ternary complexwith eNOS and Akt. Dynamin-2 regulates eNOSactivity through the binding of its proline-richdomain to the FAD domain of eNOS, promotingelectron transfer between the bound flavins of thereductase domain and increasing NO production(Cao et al., 2003; Garcia-Cardena et al., 1998). Anovel mechanism of eNOS activation and NO pro-duction in endothelial cells involves caveolae-mediated endocytosis induced by 60-kDa albumin-binding glycoprotein gp60 (Maniatis et al., 2006).Inhibition of endocytosis resulted in the markedimpairment of NO production. eNOS activity in-duced by gp60 is mediated by Gβ activation of down-stream Src, Akt, and PI3K pathways. As caveolaeinternalization is a constitutive process in endo-thelial cells, this mechanism of NO production maybe important in regulating basal NO-dependentvasomotor tone (Maniatis et al., 2006).

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