5 (2006) 308–316www.elsevier.com/locate/vph

Vascular Pharmacology 4

Oxidative and nitrosative stress in pediatric pulmonary hypertension: Rolesof endothelin-1 and nitric oxide

Stephen M. Black a,b,⁎, Jeffrey R. Fineman c,d

a Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT, United Statesb International Heart Institute, University of Montana, Missoula, MT, United States

c Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143-0106, United Statesd Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143-0106, United States

Accepted 5 August 2006

Abstract

An increasing number of studies implicate oxidative stress in the development of endothelial dysfunction and the pathogenesis ofcardiovascular disease. Further, this oxidative stress has been shown to be associated with alterations in both the endothelin-1 (ET-1) and nitricoxide (NO) signaling pathways such that bioavailable NO is decreased and ET-1 signaling is potentiated. However, recent data, from our groupsand others, have shown that oxidative stress, ET-1, and NO are co-regulated in a complex fashion that appears to be dependent on the cellularlevels of each species. Thus, when ROS levels are transiently elevated, NO signaling is potentiated through transcriptional, post-transcriptional,and post-translational mechanisms. However, in pediatric pulmonary hypertensive disorders, when reactive oxygen species (ROS) increases aresustained by ET-1 mediated activation of smooth muscle cell ETA subtype receptors, NOS gene expression and NO signaling are reduced. Further,increases in oxidative stress can stimulate both the expression of the ET-1 gene and the secretion of the ET-1 peptide. Finally, the addition ofexogenous NO, and increasingly utilized therapy for pulmonary hypertension, can also lead to increases ROS generation via the activation of ROSgenerating enzymes and through the induction of mitochondrial dysfunction. Thus, this manuscript will review the available data regarding theinteraction of oxidative and nitrosative stress, endothelial dysfunction, and its role in the pathophysiology of pediatric pulmonary hypertension. Inaddition, we will suggest avenues of both basic and clinical research that will be important to develop novel pulmonary hypertension treatment andprevention strategies, and resolve some of the remaining clinical issues regarding the use of NO augmentation.© 2006 Elsevier Inc. All rights reserved.

Keywords: Pulmonary hypertension; Reactive oxygen species; Nitrosylation; Gene expression; Cell signaling

1. Endothelin-1 and nitric oxide: key regulators ofpulmonary vascular resistance

ET-1, a 21 amino acid polypeptide produced by vascularendothelial cells, has potent vasoactive properties (Yanagisawaet al., 1988). The gene for human ET-1 is located onchromosome 6 and is translated to a 203-amino acid peptideprecursor (preproET-1), which is then cleaved to formproendothelin-1. Proendothelin, big ET-1, is then cleaved by amembrane bound metalloprotein converting enzyme (Endothe-lin Converting Enzyme-1, ECE-1) into its functional form.

⁎ Corresponding author. International Heart Institute, St. Patrick Hospital, 554W Broadway, Missoula, MT 59802, United States.

E-mail address: stephen.black@umontana.edu (S.M. Black).

1537-1891/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.vph.2006.08.005

ECE-1 exists in two isoforms, ECE-1α and ECE-1β, with ECE-1α considered to be the most biologically important (Shimadaet al., 1995). The hemodynamic effects of ET-1 are mediated byat least two distinctive receptor populations, ETA and ETB, thedensities of which are different depending on the vascular bedstudied. The ETA receptors are located on vascular smoothmuscle cells and mediate vasoconstriction, whereas the ETB

receptors are located on endothelial cells and mediatevasodilation (Arai et al., 1990; Sakurai et al., 1990; Wonget al., 1995). In addition, a second subpopulation of ETB re-ceptors is located on smooth muscle cells and mediates va-soconstriction (Shetty et al., 1993). The vasodilating effects ofET-1 are associated with the release of NO and potassiumchannel activation (Bradley et al., 1990; Cassin et al., 1991;Wong et al., 1993; Wong et al., 1995). The vasoconstricting

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effects of ET-1 are associated with phospholipase activation, thehydrolysis of phosphoinositol to inositol 1,4,5-triphosphate anddiacylglycerol, and the subsequent release of Ca2+ (La andReid, 1995). In vivo studies on the pulmonary circulation havedemonstrated that exogenous ET-1 can produce a varied hemo-dynamic response that is dependent on a variety of factors,including species, baseline pulmonary vascular tone, integrityof the vascular endothelium, alterations in receptor densities andreceptor binding, and age (Bradley et al., 1990; Cassin et al.,1991; Wong et al., 1993). In addition to endothelial cellproduction, ET-1 is produced by a variety of cell types withinthe lung, including vascular and airway smooth muscle cellsand airway epithelial cells (Fagan et al., 2001). Lastly, inaddition to its vasoactive properties, ET-1 is mitogenic forpulmonary arterial smooth muscle cells via ETA receptor-mediated superoxide generation, and therefore may participatein vascular remodeling (Wedgwood et al., 2001a). Although notwell delineated, regulation of ET-1 production appears to occurat the transcriptional level of both preproET-1 and ECE-1. Invitro studies demonstrate that preproET-1 mRNA expression isincreased in endothelial cells exposed to a variety of stimuli,which include growth factors, cytokines, and vasoactivesubstances (Kurihara et al., 1989; Imai et al., 1992; Marsdenand Brenner, 1992; Matsuura et al., 1997). In addition, ECE-1mRNA levels may be increased by exposure to growth factorsand vascular injury (Macarthur et al., 1994; Teerlink et al.,1994; Minamino et al., 1997). Other potential regulators ofplasma and tissue ET-1 levels include rapid ET-1 release fromintracellular secretory granules, alterations in ECE-1 activity,and alterations in ET-1 clearance, which appears to be mediatedin part by ETB receptors (Kuchan and Frangos, 1993; Fukurodaet al., 1994; Mitsutomi et al., 1999). In vivo data demonstrate apostnatal alteration in the hemodynamic effect of exogenousET-1 from pulmonary vasodilation to vasoconstriction (Bradleyet al., 1990; Cassin et al., 1991; Wong et al., 1993, 1994; Ivyet al., 1994, 2000). This is associated with developmentalalterations in ET-1 receptor densities (Hislop et al., 1995).Several lines of evidence suggest that ROS can also regulatecellular levels of ET-1 and mediate its secretion (Lopez-Ongilet al., 2000; Cheng et al., 2001; Wedgwood and Black, 2003b).Lastly, conflicting data suggests that NO may regulate ET-1secretion. For example, in vitro, endothelial cell mono-culturestudies demonstrate that NO decreases ET-1 signaling (Bou-langer and Luscher, 1990; Kourembanas et al., 1993; Smithet al., 2002; Kelly et al., 2004). However, contrary to thesepublished in vitro studies, we have demonstrated in vivo, thatexogenous inhaled NO increases plasma ET-1 levels (McMul-lan et al., 2001). These findings are consistent with humanreports (Pearl et al., 2002). Lastly, limited in vitro data suggestthat the modality of NO augmentation (endogenous vs.exogenous) may differentially effect ET-1 secretion (Mitsutomiet al., 1999).

NO is an endothelium-derived relaxing factor synthesized bythe oxidation of the guanidino nitrogen moiety of L-argininefollowing activation of nitric oxide synthase (NOS) (Palmeret al., 1988). Three isoforms of NOS are known. Constitutiveforms are present in endothelial cells and neurons, and a third

inducible isoform is present in macrophages (Lamas et al.,1992; Lyons et al., 1992; Sessa et al., 1993). After certainvascular stimuli, such as flow and the receptor binding ofspecific vasodilators, NO is synthesized and released from theendothelial cell by the activation of endothelial NOS (eNOS)(Rubanyi et al., 1986; Mulsch et al., 1989). Once released fromendothelial cells, NO diffuses into vascular smooth muscle cellsand activates soluble guanylate cyclase (sGC), a heterodimerwith α1 and β1 subunits which catalyzes the production ofguanosine-3′,5′-cyclic monophosphate (cGMP) from guano-sine-5′-triphosphate (GMP). cGMP induces vascular smoothmuscle relaxation through activation of a cGMP-dependentprotein kinase (Ignarro et al., 1986; Kamisaki et al., 1986;Murad, 1986). Cyclic nucleotide phosphodiesterases (PDE)regulate intracellular levels of cGMP by catalyzing cGMP toGMP (Beavo, 1995). Increasing data suggest that endothelium-derived NO (and the resulting cGMP) is an important mediatorof resting pulmonary vascular tone, a modulator of pulmonaryvasoconstriction, and an inhibitor of platelet aggregation andsmooth muscle mitogenesis (Brashers et al., 1988; Garg andHassid, 1989; Fineman et al., 1995).

Finally, a physiological antagonism between the NO-cGMPand ET-1 cascades has been well demonstrated in humanvascular tissues, cultured vascular smooth muscle cells, andleukocytes (Luscher et al., 1990; Lopez Farre et al., 1991;Okishio et al., 1992; Vanhoutte, 2000). Published data alsosuggest that NO and ET-1 participate in the regulation of eachother through an autocrine feedback loop. For example, ET-1stimulates eNOS activity via ETB receptor activation, whileNO-cGMP production increases ETA receptors in vascularsmooth muscle cells and inhibits ET-1 secretion and gene ex-pression in vascular endothelial cells (Boulanger and Luscher,1990; Kourembanas et al., 1993; Redmond et al., 1996; Kellyet al., 2004). Changes in the balance between NO generationand/or response, and ET-1 production and/or response have alsobeen described in a variety of cardiovascular diseases suchas PPHN as well as systemic hypertension, coronary arterydisease, and congestive heart failure (Rossi et al., 2001).

2. Endothelin-1 and oxidative stress

An increasing number of studies implicate oxidative stress inthe pathogenesis of cardiovascular disease and the developmentof endothelial dysfunction (Katusic, 1996; Cai and Harrison,2000). Many ROS possess unpaired electrons and are thus freeradicals. These include the superoxide anion, the hydroxylradical, NO, and certain oxidized lipids. Other ROS such ashydrogen peroxide, peroxynitrite, and hypochorous acid, arenot free radicals but are oxidizing molecules that can contributeto the overall oxidative stress. ROS can react with all biologicalmacromolecules (lipids, proteins, nucleic acids and carbohy-drates). The endothelium is a source of oxygen-derived freeradical production, especially the superoxide (O2

·−) anion(Matsubara and Ziff, 1986b,a). Sources of O2

·− productioninclude lipoxygenase, cyclo-oxygenase, xanthine oxidase, nitricoxide synthase, and NADPH oxidase (Matsubara and Ziff,1986a). However, it is generally recognized that NADPH

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oxidase is the predominant source of ROS in the vasculature(Cai et al., 2003). Several studies have demonstrated that ET-1can induce ROS formation. This increase has been observed in anumber of different situations including traumatic brain injury(Kasemsri and Armstead, 1997), in ET-1 mediated monocyteactivation (Huribal et al., 1994), and in alveolar macrophagesexposed to ET-1 (Kojima et al., 1996). Our recent studies havealso found that the vascular remodeling associated with thedevelopment of PPHN in the lamb ductal ligation model is alsoassociated with increased production of ROS (Brennan et al.,2003a; Wedgwood et al., 2005) and ET-1 (Black et al., 1998). Inaddition, recent reports indicate ROS, such as O2

·− and hydrogenperoxide (H2O2) are capable of stimulating vascular SMCproliferation (Rao and Berk, 1992; Sundaresan et al., 1995). Thelink between ET-1, oxidative stress, and SMC growth isimportant as studies have demonstrated that the proliferation ofvascular smooth muscle cells (SMC) contributes to thepathophysiology of pulmonary-, and systemic-hypertension,atherosclerosis, coronary artery restenosis after angioplasty andstent placement (Ross, 1993). In contrast recent reports haveshown that treatment of vascular SMCs with anti-oxidants (Tsaiet al., 1996) or over-expression of catalase from an adenoviralvector reduced viability and induced apoptosis in vascularsmooth muscle cells (Brown et al., 1999). Several lines ofevidence also suggest that there is a reciprocal relationshipbetween oxidative stress and ET-1 regulation such that ROS canregulate cellular levels of ET-1 and mediate its secretion(Lopez-Ongil et al., 2000; Cheng et al., 2001; Wedgwood andBlack, 2003b).

3. Interplay between oxidative and nitrosative stress

An emerging concept in signal transduction is that ROS canact as intracellular messengers (Griendling et al., 2000). Whilethe modality of signal transduction by ROS is far fromcompletely understood, it is believed that ROS are necessarycomponents in transducing the mitogenic effects of a number ofgrowth factors. Indeed, it is now becoming apparent that H2O2

has an increasingly important and variable role in mammaliancell physiology. Under normal physiological conditions, mostintracellular H2O2 is formed by the dismutation of O2

·−, abyproduct of incomplete reduction of O2 during the process ofoxidative phosphorylation. Evidence indicates that H2O2 canfunction as a second messenger in multiple signal transductionpathways, in a manner not dissimilar to nitric oxide (NO). Anumber of different cell types have been shown to produce O2

·−

and H2O2 in response to extracellular stimuli. Situations whereloss of regulation of H2O2 occurs, either by excessive productionor compromised ability to dispose of H2O2, are believed to play acentral role in disease and aging. Indeed, we recently reportedthat acute exposure to high levels of H2O2 induced an apoptoticresponse in pulmonary endothelial cells (Wedgwood and Black,2005). When H2O2 reaches potentially toxic levels, mostattention to date has focused on the resulting oxidative damageto proteins, lipid, and nucleic acids. In addition, our recent dataindicate that ET-1 can induce H2O2 production in PASMCwhichcan feedback and inhibit both NOS activity and expression in

PAEC (Wedgwood and Black, 2005). Similar effects could beproduced by direct addition of 100 μM H2O2 (Wedgwood andBlack, 2005). However, the mechanisms by which H2O2

regulates eNOS are complex and incompletely understood. Forexample, Drummond et al. (2000) have found anH2O2-mediatedconcentration- and time-dependent increase in eNOS expressionrelated both to increases in eNOS transcription and mRNAstability. We found that H2O2 increased eNOS expression atlower concentrations in EC isolated from fetal lambs (FPAEC),but not at the higher H2O2 concentrations used in Drummond'sstudy. Although these data appear to conflict it is possible thatthe differences in anti-oxidant capacity we have observed, whereBAEC exhibited a 2-fold higher catalase activity than FPAEC,may account for the observed responses to H2O2. In ECs fromthe fetal pulmonary circulation, which would normally encoun-ter blood with low oxygen content, the reduced anti-oxidantactivity may render the cells more sensitive to ROS. In cells fromthe adult aorta, which contact blood with high oxygen content,the increased anti-oxidant activity may prevent oxidativedamage. After birth and continuing into adulthood the pulmo-nary system is exposed to higher concentrations of oxygen thanother organs, whichmay render it more vulnerable to free-radicalmediated injury (Asikainen et al., 1998). Defensemechanisms oflung, and other, tissues against ROS-mediated injury includeboth small molecular weight antioxidants (for example vitaminsC, E, reduced GSH, etc.) and antioxidant enzymes. Theseinclude CuZn- and Mn-SOD, glutathione peroxidase, andcatalase. There have been several studies investigating thedevelopmental expression of these enzymes in lung and othertissues (Jenkinson et al., 1986; Rickett and Kelly, 1990; Clerchand Massaro, 1992a,b; McElroy et al., 1992; Yuan et al., 1996;Asikainen et al., 1998; Pietarinen-Runtti et al., 1998). Theimportance of catalase in the lung is poorly defined (Asikainenet al., 1998). However, studies on the developmental regulationof anti-oxidant enzymes have been carried out from a number ofspecies including rabbit (Frank and Groseclose, 1984), rat(Clerch and Massaro, 1992b), guinea pig (Rickett and Kelly,1990; Yuan et al., 1996), and human (Asikainen et al., 1998).Although the relative changes in expression vary in each speciesit was shown that catalase increases both in expression andactivity during development of the lung. In humans it was foundthat catalase expression was around 2-fold higher in neonatesthan fetuses and 7–8-fold higher in adults (Asikainen et al.,1998). The changes in activity were found to correlate closelywith these changes in expression (Asikainen et al., 1998).Interestingly, the authors found that the developmental changesin catalase expression and activity were NOT found in the liver(Asikainen et al., 1998). Also, it should be noted that it has notbeen determined whether catalase (or other antioxidantenzymes) expression or activity is altered in clinical conditionssuch as endothelial dysfunction.

4. Pediatric pulmonary hypertension and endothelialdysfunction

Pulmonary hypertensive disorders are a significant source ofmorbidity and mortality in the pediatric population. In neonates,

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the most common etiology results from a failure to undergo thenormal fall in pulmonary vascular resistance at birth (persistentpulmonary hypertension of the newborn, PPHN), with anincidence of ∼1 per 1000 live births. However, otherpulmonary abnormalities, such as congenital diaphragmatichernia, respiratory distress syndrome, and bronchopulmonarydysplasia, may also result in neonatal pulmonary hypertension.Beyond the neonatal period, the majority of pediatricpulmonary hypertensive disorders are associated with congen-ital heart defects. Other, less common causes of pediatricpulmonary vascular disease include primary (idiopathic)pulmonary hypertension, hypoxia-induced pulmonary vasculardisease, rheumatologic disorders, sickle cell disease, portalhypertension, chronic thromboembolic disease, HIV disease,and drug-toxin induced disease.

Increasing evidence suggests that endothelial injury and theresulting alteration in the balance of these and other vasoactivesubstances has a significant role in the development of pulmo-nary hypertension and increased vascular reactivity (Rabino-vitch et al., 1986; Dinh Xuan et al., 1990; Celermajer et al.,1993). Support for this hypothesis is strengthened by observa-tions that endothelial injury precedes pulmonary hypertensionand its associated vascular remodeling in several animal modelsof pulmonary hypertension (Meyrick et al., 1980; Adnot et al.,1991). In humans, endothelial dysfunction, including histolog-ical abnormalities of the endothelium, impairment of endothe-lium-dependent pulmonary vasodilation, and increased plasmaET-1 concentrations have been described in children withcongenital heart defects and pulmonary hypertension before thedevelopment of significant vascular remodeling (Rabinovitchet al., 1986; Yoshibayashi et al., 1991; Celermajer et al., 1993).In addition, neonates with PPHN, and adults with advancedpulmonary vascular disease have evidence of endothelialdysfunction, impairment of endothelium-dependent pulmonaryvasodilation, increased plasma ET-1 concentrations and de-creased prostacyclin production (Dinh Xuan et al., 1990;Christman et al., 1992; Giaid et al., 1993; Giaid and Saleh,1995).

There is also increasing evidence that alteration in theproduction and/or ability to scavenge ROS participate in theendothelial dysfunction associated with pulmonary hyperten-sion. We have shown that both O2

·− (Brennan et al., 2003a) andH2O2 (Wedgwood et al., 2005) are increased in an ovine ductalligation model of PPHN while our data also suggest that anti-oxidants increase NO-mediated signaling in these lambs(Brennan et al., 2003a; Wedgwood et al., 2005). In addition,our data suggest that a sustained increase in H2O2 levels mayexplain the coordinated decrease in vasodilator genes observedin PPHN (Wedgwood and Black, 2005; Wedgwood et al., 2005;Black et al., 1998).

The mechanism of injury to the vascular endothelium isunclear, but is likely multi-factorial, and in part dependent uponthe etiology of the pulmonary hypertension. For example, inneonates with PPHN and children with congenital heart diseaseand increased pulmonary blood flow, the initiating endothelialinjury is likely mediated by increased shear stress. However,once pulmonary arterial pressure is elevated, shear stress-

mediated endothelial injury appears to promote the progressionof the disease, independent of the underlying etiology.Following an initial endothelial injury, smooth muscle prolif-eration and progressive structural remodeling occurs. Regard-less of the etiology, advanced disease is characterized by medialhypertrophy, intimal hyperplasia, angiomatoid formation, in situthrombi, and eventual vascular obliteration. Untreated, thesestructural changes progress to the point of becoming function-ally “fixed,” or irreversible. An important goal of therapy is toavoid acute pulmonary vasoconstriction, halt the progression ofvascular remodeling, and reverse the early vascular remodelingif possible. To this end, augmentation of the NO-cGMP cascadeis an increasingly utilized therapeutic approach.

5. Nitric oxide-cGMP augmentation

NO donor compounds (i.e. nitroglycerin and sodiumnitroprusside) have had a major role in the treatment of vasculardisorders for decades. Following the more recent discoveries ofNO biology, the use of free inhalation NO gas has emerged as animportant treatment for a variety of pulmonary vascular andparenchymal diseases. Currently, its major use is as a selectivepulmonary vasodilator, secondary to rapid inactivation byhemoglobin, in newborns with persistent pulmonary hyperten-sion, and infants, children, and adults with peri-operativepulmonary hypertension (Kinsella et al., 1997; Roberts et al.,1997). In addition, because of its inhalational route, inhaled NOimproves ventilation–perfusion matching and is being utilizedto improve oxygenation in patients with acute lung injury (Dayet al., 1997; Dellinger et al., 1998; Dobyns et al., 1999). Lastly,because of its vasodilatory, anti-platelet, and anti-mitogeniceffects, inhaled NO is also being delivered chronically inclinical trials for advanced pulmonary vascular disorders andthe prevention of chronic lung disease in premature infants(Schreiber et al., 2003). Although preliminary results areencouraging, increasing experience has demonstrated unpre-dictable and non-sustained responses to inhaled NO, and aclinically significant rapid increase in pulmonary vascularresistance upon its acute withdrawal (Miller et al., 1995; Atzet al., 1996; Lavoie et al., 1996; Cueto et al., 1997). Recent datasuggest that alterations in endogenous endothelial functionmediate this rebound pulmonary hypertension, but its mechan-isms are unclear (Sheehy et al., 1998; Wedgwood et al., 2001b).These clinical observations and recent laboratory data suggestthat exogenously administered inhaled NO may alter endoge-nous pulmonary endothelial function (Sheehy et al., 1998;Black et al., 1999b; McMullan et al., 2001; Wedgwood et al.,2001b). For example, both in vitro and in vivo data demonstratethat exogenous NO exposure alters the endogenous NO-cGMPand endothelin (ET)-1 cascades (Black et al., 1999b; Wedg-wood et al., 2001b). Both in vitro and in vivo studies dem-onstrate that exogenous NO can decrease endogenous eNOSactivity, independent of changes in gene expression (Sheehyet al., 1998; Black et al., 1999b; McMullan et al., 2001;Wedgwood et al., 2001b). In addition, recent studies from ourgroups have demonstrated an increase in plasma ET-1 levelsduring inhaled NO therapy, and suggest a role for ET-1 in the

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pulmonary vasoconstriction associated with the withdrawal ofNO therapy. Moreover, these studies suggest a link betweenETA-receptor activation and decreased eNOS activity, as ETA-receptor antagonism was shown to block the decrease in eNOSactivity observed during inhaled NO exposure (McMullan et al.,2001).

Our recent in vitro studies have also demonstrated a role forO2·− in the link between increases in ET-1 and decreases in

eNOS activity during NO exposure (Wedgwood et al., 2001b).To summarize, our in vitro data indicate that exogenous NO canactivate two signaling pathways. The first results in ETAreceptor-mediated increases in O2

·− production, resulting in theformation of peroxynitrite and subsequent nitration andinactivation of eNOS (Wedgwood et al., 2001b). The secondinvolves the NO-dependent activation of p21ras. Studies fromLander et al. (1997) have localized the molecular interactionbetween NO and p21ras to amino acid cysteine 118. It has beendemonstrated that NO induces a nitrosylation event on thecysteine 118 thiol residue triggering GDP/GTP exchange(Lander et al., 1996, 1997). This leads to an increased popu-lation of active Ras-GTP (Lander et al., 1996). Once in this GTPbound state Ras is then available to interact with downstreamtargets leading to a diverse array of biological responses such ascell growth, cell differentiation, programmed cell death, etc. Inaddition to these biological processes it has been postulated thatNO stimulated guanine-nucleotide exchange may be animportant physiological mechanism for the activation of signaltransduction pathways mediated by Ras that are redox sensitive(Mott et al., 1997). However, it is still far from clear how cellsmay achieve selectivity while utilizing this type of redoxsignaling. Indeed our in vitro data indicate that NO-mediatedactivation of p21ras in endothelial cells leads to an increase inSer1177 phosphorylation that is associated with an increase inO2·− generation from eNOS (Brennan et al., 2003b). In addition,

work from us, and others, have shown that this increase in O2·−

generation from eNOS may be also be dependent on peroxyni-trite-mediated nitration of eNOS (Brennan et al., 2002a; Zou et al.,2002). However, it is likely that other vascular enzyme systemsare involved in the generation of ROS in response to exogenousNO. For example, our previous studies have suggested that thexanthine oxidase system can be activated by NO donors (Sheehyet al., 1998) while the activation of the NADPH oxidase complexhas been shown to be activated by nitrogylcerin (Munzel et al.,1995) and Spermine NONOate (Brennan et al., 2002b). Finally,we have shown that the induction of apoptosis induced by NOdonors in VSMCs is associated with a loss of mitochondrialmembrane potential and a transient increase in ROS (Wedgwoodand Black, 2003a).

Finally, it is becoming clear that nitrosative stress can alsoregulate NOS activity. We have recently shown that NO donorsdisrupt the active eNOS dimeric complex via an S-nitrosylationof the zinc-tetrathiolate cluster (Ravi et al., 2004) while Zouet al. (2002) have shown a similar effect on eNOS that ismediated by peroxynitrite. A disruption of the iNOS dimermediated by S-nitrosylation has also been recently observed(Mitchell et al., 2005) suggesting that this could be a commonmechanism by which NOS can be regulated. It has also been

suggested that S-nitrosylation could serve to keep NOS in aninactive form until stimulated as S-nitrosylation was recentlyshown to decrease when VEGF was added to EC (Erwin et al.,2005). It remains to be determined whether ROS can alsoinduce an inhibition of NOS through a similar disruption of theNOS dimer although we have shown that ET-1 inducedincreases in H2O2 can reduce eNOS activity (Wedgwood andBlack, 2005). This is in contrast to other studies who haveshown that H2O2 can increase NOS activity (Drummond et al.,2000) through an increase in Ser1177 phosphorylation mediatedby an activation of a Src/PI3 kinase/Akt signaling pathway(Drummond et al., 2000; Thomas et al., 2002).

6. Summary and future directions

As detailed above previous studies have implicated theoxidative stress mediated by O2

·− and H2O2 in the pathophys-iology of a number of cardiovascular disorders. Furthermore,we have previously shown that antioxidants attenuate FPASMCgrowth and at high doses induce apoptosis in vitro (Wedgwoodand Black, 2003a) and increase NO-signaling in vivo (Brennanet al., 2003a; Wedgwood et al., 2005), suggesting thatantioxidant therapy may represent a useful treatment strategyfor patients with pulmonary hypertension. However, a recentstudy has shown that ROS levels are increased in ECs exposedto VEGF and that this correlated with a mitogenic response(Colavitti et al., 2002). This suggests that ECs also utilize ROSas signaling molecules. Thus, therapies based on high doseantioxidants may have adverse effects on EC that could poten-tiate the underlying endothelial dysfunction seen in PPHN andother vascular disorders. Thus, treatment strategies may requirecell type specific targeting.

As described above a number of cardiovascular disordersare associated with decreased levels of bioavailable NO andtherapies based on augmenting NO levels are now employed totreat a number of these disease states. However, there are anumber of unresolved issues with the use of inhaled NO and NOdonors. For example, in children with PPHN treated with inhaledNO only around 30–50% have beneficial effects but still canhave the rebound pulmonary hypertension associated with acuteNO withdrawal. Thus, we need to develop ways to identify non-responders prior to implementing inhaledNO therapy. Related tothis issue is what doses of inhaled NO or NO donors should beemployed and shouldwe be using pharmacologic or replacementlevels of NO? This is an important issue as it is becoming moreapparent that NO is not without associated toxicities. However,this issue is hampered by a lack of understanding regarding whatare “normal”NO levels and what are the age and sex differencesthat need to be taken into account. Indeed, due to the underlyingissues with NO donors as therapies, new modalities are beingdeveloped to target endogenous NO-cGMP augmentation forpulmonary hypertensive disorders that are not dependent onexogenous NO. These include L-arginine for pulmonaryhypertension associated with sickle cell disease, sildenafil(a phosphodiesterase 5 inhibitor) for peri-operative and chronicpulmonary hypertensive disorders, and anti-oxidant treatmentsfor vascular disease associated with heart failure (Stephens et al.,

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1996; Irukayama-Tomobe et al., 2000; Watanabe et al., 2002;Griendling and FitzGerald, 2003;Michelakis et al., 2003;Morriset al., 2003; Stocker et al., 2003). These therapies are novel andclinical data is sparse. Moreover, their effects on endogenousendothelial function have not been thoroughly investigated.

Acknowledgments

This research was supported in part by grants HL60190 (toSMB), HL67841 (to SMB), HL72123 (to SMB), HL70061 (toSMB), and HL61284 (JRF) from the National Institutes ofHealth, and 0330292Z from the American Heart AssociationPacific Mountain Affiliates (to SMB).

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