Early determinants of pulmonary vascular remodeling

Schreiber, Thomas Kietzmann, Ian Adatia, John Hess and Stephen M. BlackSohrab Fratz, Jeffrey R. Fineman, Agnes Görlach, Shruti Sharma, Peter Oishi, Christian

Congenital Heart DiseaseEarly Determinants of Pulmonary Vascular Remodeling in Animal Models of Complex

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2011 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.110.978528

2011;123:916-923Circulation.

http://circ.ahajournals.org/content/123/8/916World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circ.ahajournals.org//subscriptions/

is online at: Circulation Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answer this process is available in the

click Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located,

can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialCirculationin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:

by guest on April 7, 2014http://circ.ahajournals.org/Downloaded from by guest on April 7, 2014http://circ.ahajournals.org/Downloaded from

http://circ.ahajournals.org/content/123/8/916

http://circ.ahajournals.org/content/123/8/916

http://www.ahajournals.org/site/rights/

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://www.lww.com/reprints



http://circ.ahajournals.org/




Basic Concepts in Heart Failure

Early Determinants of Pulmonary Vascular Remodeling inAnimal Models of Complex Congenital Heart DiseaseSohrab Fratz, MD*; Jeffrey R. Fineman, MD*; Agnes Gorlach, MD; Shruti Sharma, PhD;

Peter Oishi, MD; Christian Schreiber, MD; Thomas Kietzmann, PhD; Ian Adatia, MD;John Hess, MD; Stephen M. Black, PhD

In this article, we review the current state of knowledgeabout early changes in the pulmonary vasculature that

result from persistent systemic-to–pulmonary arterial shunt-ing in newborn lambs. Data generated in this model systemmay be important in children with various forms of congen-ital heart disease (CHD), but perhaps most so in those withsingle-ventricle anomalies. Children born with a functionalsingle ventricle (eg, hypoplastic left heart syndrome, tricuspidatresia, or pulmonary atresia) represent a subgroup of patientswith CHD with the poorest outcome, with 5-year mortalityrates up to 50%.1 Although functionally single-ventricle heartdisease comprises many structural variants, a shared physio-logical feature is that both systemic and pulmonary circula-tions are supplied in parallel by a functionally single pumpingchamber. Immediately after birth, the pulmonary vasculatureof these infants is exposed to abnormal conditions, such asincreased flow or pressure.2–4 Over time, if these abnormalforces are not modified, they can lead to progressive func-tional and morphological abnormalities in the pulmonaryvasculature that are characterized by altered reactivity, in-creased resistance, and structural alterations (remodeling).3

Clinically, these abnormalities can have an important impacton surgical options and perioperative outcome. The currentapproach to neonates with a functional single ventricle is toestablish a controlled, low-pressure source of pulmonaryblood flow that facilitates systemic oxygen delivery sufficientto permit somatic growth and development without excessiveventricular volume loading, typically by means of a surgicalsystemic-to–pulmonary artery or ventricle-to–pulmonary ar-tery conduit or by restricting the main or branch pulmonaryarteries.2 The pulmonary-to-systemic blood flow balance canbe difficult to achieve, and the infant runs the gauntletbetween pulmonary blood flow that is too low (resulting inchronic hypoxemia and the potential consequences thereof)or too high (potentially leading to chronic heart failure,compromised systemic blood flow, and abnormal pulmonary

vascular remodeling). Because subsequent stages of surgicalpalliation (bidirectional or partial cavopulmonary connection,followed by Fontan-type surgery or total cavopulmonaryconnection) result in passive pulmonary blood flow in theabsence of a pumping ventricle, a normal low pulmonaryvascular resistance is critical for maintenance of low centralvenous pressure, overall health, and long-term survival.5 Theunderlying mechanisms by which some patients with single-ventricle physiology experience increased pulmonary vascu-lar resistance that results in failed surgical palliation whereasothers do not remain unclear. We speculate that these differ-ences in outcome are related to early, clinically undetectablepulmonary vascular abnormalities. For example, age at sur-gery correlates with increased transpulmonary gradient post-operatively in infants with a functional single ventricleundergoing and surviving partial cavopulmonary connection(Figure 1). This may be because of the longer duration overwhich the pulmonary vasculature was exposed to increasedflow. Figure 1 suggests that a relatively large surgical shuntwith relatively high pulmonary blood flow (thereby allowingfor an older age at partial cavopulmonary connection) maylead to a subtly higher transpulmonary gradient (whichsuggests higher pulmonary vascular resistance) that may bedetrimental in the long-term. This suggestion remains spec-ulative because it is unknown whether subtly increasedresistance leads to demonstrably worse outcome. However, itis likely that there are subtle differences in signaling path-ways or gene-expression variations between patients. Thesesignaling pathways, acted on by the increases in pulmonaryblood flow, can then lead to subtle differences in pulmonaryvascular resistance that dramatically affect the surgical out-come. The underlying pathways in these newborns withsingle-ventricle heart disease are only just beginning to beresolved. In this review, we will focus on the work over thelast decade that has begun to elucidate the molecular path-ways that appear to be important in the initial pulmonary

From the Department of Pediatric Cardiology and Congenital Heart Disease (S.F., A.G., J.H.) and Department of Cardiovascular Surgery (C.S.),Deutsches Herzzentrum Munchen, Klinik an der Technischen Universitat Munchen, Munich, Germany; Department of Pediatrics (J.R.F., P.O.) and theCardiovascular Research Institute (J.R.F.), University of California, San Francisco, CA; Pulmonary Vascular Disease Program (S.S., S.M.B.), VascularBiology Center, Medical College of Georgia, Augusta, GA; Department of Biochemistry (T.K.), Technical University of Kaiserslautern, Kaiserslautern,Germany; and the Department of Pediatrics (I.A.), University of Alberta, Edmonton, Canada.

*Drs Fratz and Fineman contributed equally to this article.Correspondence to Stephen M. Black, PhD, Vascular Biology Center, 1459 Laney Walker Blvd, CB3211B, Medical College of Georgia, Augusta, GA

30912. E-mail [email protected](Circulation. 2011;123:916-923.)© 2011 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.110.978528

916 by guest on April 7, 2014http://circ.ahajournals.org/Downloaded from



vascular remodeling in newborns with single-ventricle heartdisease.

Lamb Model of Neonatal IncreasedPulmonary Blood Flow

Although many animal models have been used to studyincreased blood flow or pressure on the pulmonary vascula-ture, most models use older or adult animals.6 Therefore,these models neglect the initial postnatal effects of increasedblood flow or pressure on the developing pulmonary vascu-lature during the natural course of postnatal transition fromfetal to adult pulmonary blood flow. A model to study theinitial onset of pulmonary vascular remodeling associatedwith single-ventricle physiology needs to have a large aorto-pulmonary shunt from birth so that the natural course ofpostnatal transition from fetal to adult pulmonary blood flowcan be considered in the pathophysiology. To the best of ourknowledge, the only existing animal model that can be usedto study the initial onset of neonatal pulmonary vascularremodeling associated with single-ventricle physiology is theneonatal lamb model with an aortopulmonary shunt placed inutero (Figure 2).7 This model does have its limitations as atrue mimic of single-ventricle physiology. The shunt modelallows studies into the effects of increased pulmonary bloodflow and pressure on pulmonary vascular signaling and geneexpression; however, it may not take into account the influ-ence of ventricular compliance or function that likely plays arole in the pulmonary vascular remodeling observed inneonates with single-ventricle physiology. Nevertheless, it isthe best model available for these types of investigations, andover the last decade, this model has been used extensively tostudy the early sequential evolution of pulmonary vascular

remodeling mimicking the pathophysiologically relevant el-ements of neonatal pulmonary blood flow in single-ventricleheart disease. In the following sections, we will brieflydescribe the current knowledge regarding the well-describednitric oxide (NO) and endothelin-1 (ET-1) signaling path-ways, as well as newly emerging pathways that may havepotential for therapeutic intervention.

Established Signaling PathwaysThe NO-cGMP Signaling CascadeNO production by the vascular endothelium is integral to themaintenance of the low-resistance state of the pulmonaryvasculature, and dynamic alterations in NO production mod-ulate vascular relaxation and constriction in response tovarious stimuli. Once formed, NO diffuses into vascularsmooth muscle cells, where it activates soluble guanylate

Figure 1. Retrospective analysis of 69 consecutive patients younger than 15 months (subgroup from study by Cleuziou et al4) withsingle-ventricle physiology who underwent and survived partial cavopulmonary connection (PCPC). The x-axis depicts age at PCPC, inmonths; y-axis depicts transpulmonary gradient, defined as left atrial pressure minus pulmonary artery pressure during heart catheter-ization before total cavopulmonary connection (TCPC) surgery.

Figure 2. Representative hematoxylin-and-eosin stains of pe-ripheral lung fixed in 4% paraformaldehyde and sectioned at7 �m from 4-week control (left) and shunt (right) lambs. Notethe increase in medial thickness in the shunt lambs. Magnifica-tion �20.

Fratz et al Vascular Remodeling in CHD 917

by guest on April 7, 2014http://circ.ahajournals.org/Downloaded from



cyclase, which leads to increases in the second messenger,cGMP. The resulting activation of cGMP-dependent proteinkinase in the pulmonary vascular smooth muscle layer leadsto a decrease in intracellular calcium and relaxation of thevessel.8 Extensive temporal investigations in the shunt modelover the first 2 months of life have shown that there is acomplex pattern of regulation in the NO-cGMP signalingcascade that occurs both before and after overt alterationsin endothelial function or vascular remodeling. As shownin Table 1, over the first 2 months of life, there is aprogressive loss of NO signaling. Loss of NO signaling isalso found in humans with advanced forms of pulmonaryvascular remodeling.9,10 Decreases in NO metabolites havealso been found in children with CHD and increasedpulmonary blood flow,11,12 as well as after cardiopulmo-nary bypass.13,14 Interestingly, a genetic polymorphism(894G3T) has also been identified with CHD.15 Thispolymorphism has been associated with decreased NOgeneration in humans.16,17 Accordingly, inhalation of NOgas18 and therapies aimed at enhancing NO signaling havealready become a common treatment strategy for newbornswith increased pulmonary resistance.19,20

The Endothelin SystemThe hemodynamic effects of ET-1 are mediated by at least 2distinctive receptor populations, ETA and ETB, the densitiesof which are different depending on the vascular bed studied.ETA receptors are located on vascular smooth muscle cellsand mediate vasoconstriction, whereas ETB receptors arelocated on endothelial cells and mediate vasodilation.7,21,22 Inaddition, a second subpopulation of ETB receptors is locatedon smooth muscle cells and mediates vasoconstriction.23 Thevasodilating effects of ET-1 are thought to be associated withthe release of NO and potassium channel activation.22,24,25

The vasoconstricting effects of ET-1 are associated withphospholipase activation; the hydrolysis of phosphoinositolto inositol 1,4,5-triphosphate, and diacylglycerol; and thesubsequent release of Ca2�.26 In addition to its vasoactiveproperties, ET-1 is mitogenic for pulmonary arterial smoothmuscle cells via ETA receptor–mediated superoxide genera-tion and therefore may participate in vascular remodeling.27

Patients with advanced forms of pulmonary vascular remod-eling have increased lung and circulating ET-1 levels.28

Furthermore, pulmonary blood pressure and not flow isassociated with net ET-1 production in patients with CHDand normal pulmonary vascular resistance.29,30 As with theNO-cGMP signaling pathway, shunt lambs exhibit temporalalterations in the ET-1 cascade. As shown in Table 1, within1 week of life, there is enhanced ET-1–mediated signaling inshunt lambs, whereas at 4 weeks of age, the enhancedcirculating levels of ET-1 lead to increased vasoconstrictionthrough increases in the expression of ETA receptor anddecreases in ETB receptor. By 2 months of age, expression ofthe ETB receptor has returned to control levels, but itslocalization is altered, being predominantly on the smoothmuscle cells, where it then mediates vasoconstriction. Ther-apies aimed at decreasing the vasoconstrictor and prolifera-tive effects of ET-1 are being pursued for newborns withincreased pulmonary resistance.20,31 However, the fact thatthere are changes in the expression and localization of ETreceptors in shunt lambs during the temporal progression ofboth endothelial dysfunction and vascular remodeling meansthat the correct receptor antagonist strategy (ETA versus ETB

versus dual) is still controversial. As Table 1 suggests, thebest therapeutic option may depend on where the patient is inthe disease process.

Emerging Signaling PathwaysRecent studies in shunt lambs have identified 5 signalingpathways that may represent therapeutic targets: Peroxisomeproliferator–activated receptor (PPAR)-�, arginine metabo-lism, oxidative and nitrosative stress, thrombin, and carnitine.Furthermore, microarray analyses have identified new classesof genes that appear to be associated with early pulmonaryvascular remodeling and may prove to be amenable totherapeutic interventions.

Peroxisome Proliferator–Activated ReceptorsPPARs are ligand-activated transcription factors that belongto the nuclear hormone receptor family. Recent evidence hasestablished a role for altered PPAR signaling in the develop-ment of both systemic and pulmonary vascular disease.32

Table 1. Developmental Changes in NO and ET-1 Signaling inShunt Lambs

1 Week 2 Weeks 4 Weeks 8 Weeks

NO Signaling

eNOS protein levels 7 7 1 7

Relative NOS activity 7 2 22 222

Plasma NOx levels ND 1 7 7

Lung tissue NOx levels ND 1 7 7

Nitrated eNOS ND 1 11 111

Hsp90 protein levels ND 7 7 7

eNOS bound Hsp90 ND 2 22 ND

Plasma cGMP levels 7 1 1 1

Lung tissue cGMP levels ND 1 1 1

sGC-� protein levels ND 1 1 7

sGC-� protein levels ND 7 1 7

PDE5 protein levels ND 7 1 7

PDE5 activity ND ND 1 ND

Plasma BNP levels ND 7 1 1

ET signaling

Plasma ET-1 ND — 1 ND

Immunoreactive ET-1 1 — ND ND

PreproET-1 mRNA 7 — 7 ND

PreproET-1 protein levels 7 — 7 ND

ECE-1 mRNA ND — 1 ND

ECE-1protein levels 1 — 1 ND

ETA receptor protein levels 7 — 1 1

ETB receptor protein levels 2 — 2 7

NOS indicates NO synthase; NOx, nitrogen oxide; ND, not determined; Hsp90,90-kD heat shock protein; sGC, soluble guanylate cyclase; PDE5, phosphodi-esterase type 5; BNP, brain natriuretic peptide; ET, endothelin; ECE-1,endothelin-converting enzyme-1; 7, no change; 2, mildly decreased; 22,moderately decreased; 222, severely decreased; 1, mildly increased;11, moderately increased; and 111, severely increased.

918 Circulation March 1, 2011




Recent experimental studies suggest that the PPAR-� isoformmay have a role in advanced pulmonary hypertension.33,34

PPAR-� signaling is attenuated in shunt lambs.35 A microar-ray analysis has been performed in ovine pulmonary endo-thelial cells exposed to the PPAR-� inhibitor GW9662 tomimic the loss of PPAR-� signaling. This analysis identifiedmore than 100 genes that were either upregulated or down-regulated (Table 2).35 The upregulated genes are broadlyclassified into 4 categories: Cell cycle–related genes,angiogenesis-related genes, ubiquitin-related genes, and zincfinger proteins. Furthermore, these in vitro results wereconfirmed in the shunt lamb by Western blot analysis.35

Therefore, potential targets that may be good candidates fortherapeutic intervention in patients with early neonatal pul-monary vascular remodeling associated with single-ventriclephysiology may soon be identified.

Arginine MetabolismL-Arginine bioavailability plays a key role in the generationof NO in the pulmonary vasculature. L-Arginine is activelytransported into endothelial cells through the cationic aminoacid transporter-1, where it is then used as a substrate byendothelial NO synthase (eNOS) to form NO and L-citrullineor metabolized by arginase to form urea and ornithine. Thus,eNOS and arginase are in direct competition for L-arginine. Inaddition, there resides within the caveolus a recycling path-way for L-arginine, in which the enzymes argininosuccinatesynthetase and argininosuccinate lyase recycle L-citrullineback to L-arginine. L-Arginine metabolism is disrupted inshunt lambs through a number of mechanisms.36 L-Argininerecycling is impaired owing to decreased activity of argini-nosuccinate synthetase and argininosuccinate lyase, andL-arginine catabolism is enhanced because of increases inarginase activity.36 Although expression of the cationic aminoacid transporter-1 is increased, which would enhanceL-arginine levels in the cell, the decrease in L-citrullinerecycling combined with increased L-arginine catabolismresults in a decrease in relative NO signaling.36 Thus, it islikely that the progressive loss of NO signaling in shuntlambs8 is mediated at least in part by decreases in availableL-arginine. This new description of the L-arginine–NO path-way fits well with previous studies in a rat model ofhypoxia-induced pulmonary hypertension.37 Taken together,these studies suggest that treatment strategies to enhanceL-arginine bioavailability in neonates with early pulmonaryvascular remodeling associated with single-ventricle physiol-ogy may have clinical utility.

Oxidative and Nitrosative StressExcessive production of reactive oxygen species (ROS),outstripping endogenous antioxidant defense mechanisms,has been implicated in many disease processes of the vascu-lature.38 Decreases in bioavailable NO can be related toincreases in oxidative stress. In shunt lambs, oxidative stressoccurs secondary to both increased production of ROS39 anddecreases in their scavenging.40 The source of the increasedsuperoxide is multifactorial, involving the upregulation ofvascular xanthine oxidase and NADPH oxidase and anuncoupling of eNOS.8,41,42 The mechanisms underlying theuncoupling of eNOS (ie, the consumption of NADPH be-comes uncoupled from NO synthesis) are also complex andinvolve decreases in L-arginine36 and alterations in biopterinmetabolism.39 Superoxide and other ROS have also beenshown to have a signaling function in vascular cells thatpromotes hypertrophy, proliferation, migration, matrix re-modeling, and even the formation of new vessels. Theactivation of NADPH oxidases with subsequent ROS produc-tion has been associated with increased proliferation ofpulmonary artery smooth muscle cells isolated from bothsheep27 and humans.43

Both NO and superoxide possess unpaired electrons andreact rapidly to form the reactive nitrogen species peroxyni-trite (ONOO�), which decreases NO bioavailability. In addi-tion, ONOO� can exert effects through the nitration ofprotein tyrosine residues. Tyrosine nitration, a covalent mod-ification that adds a nitro group (�NO2) to the ortho carbonof the phenolic ring, yields 3-nitrotyrosine.44 The nitration oftyrosine residues to form 3-nitrotyrosine is widely used as amarker of ONOO� formation, and recent studies have shownthat there is a temporal increase in protein nitration in thelungs of shunt lambs.8 Furthermore, eNOS itself is suscepti-ble to nitration,8 and mass spectroscopy studies have demon-strated that these nitrated residues are in regions of the proteinthat are important to its function.45 Thus, the potential ofantioxidant therapies to reduce both ROS and reactive nitro-gen species formation has been postulated for the treatment ofearly neonatal pulmonary vascular remodeling associatedwith single-ventricle physiology. However, these studieshighlight the limitation of current investigations: The lack ofeasily available means to identify modified proteins andresidues. This is a roadblock in understanding the potentialmechanistic contribution of these modifications, and it isimperative that there be a greater focus on identifying theindividual tyrosine residues targeted by nitration and theeffect these nitration events have on the structure-functionrelationship of the protein. Only when these goals have beenmet will it be possible to develop directed therapies.

ThrombinRecent studies have elucidated a novel positive-feedbackloop in which thrombin increases ROS levels via the consec-utive activation of protease activated receptor 1 (PAR1),Rac1, and p21-activated kinase.43 This also results in activa-tion of the transcription factor nuclear factor-�B, which inturn leads to activation of hypoxia-inducible factor-1�.46,47

Under these conditions, hypoxia-inducible factor-1 targetgenes, such as plasminogen activator inhibitor-1 or vascular

Table 2. Endothelial Cell Genes Regulated by PPAR-�

Functional ClassificationNo. of GenesUpregulated

No. of GenesDownregulated

Cell cycle–related genes 7 0

Angiogenesis-related genes 11 1

Ubiquitin-related genes 9 0

Zinc finger genes 17 1

Unclassified 56 18

Total 100 20





endothelial growth factor, are upregulated. The upregulationof plasminogen activator inhibitor-1, which is the primaryphysiological inhibitor of tissue plasminogen activator andurokinase plasminogen activator, can then lead to reductionsin fibrin clearance. In addition, increases in plasminogenactivator inhibitor-1 can promote pulmonary artery smoothmuscle cell proliferation. Together, these antifibrinolytic andgrowth-modulating activities have an immediate impact onpulmonary vascular remodeling, and p21-activated kinaselevels are elevated in remodeled pulmonary vessels in pa-tients with CHD and pulmonary vasculopathy.43,48 Thereappears to be a secondary feed-forward loop activated byhypoxia-inducible factor-1 that leads to further increases inthe expression of Rac1 and p21-activated kinase, producingenhanced cytoskeletal remodeling and proliferation of pul-monary artery smooth muscle cells, which further contributesto medial hypertrophy and vascular remodeling.43 As part ofthis feed-forward mechanism, ROS levels are stimulatedbecause of the increased abundance of Rac1 and p21-acti-vated kinase, which produces sustained activation of thehypoxia-inducible factor-1 pathway and increased pulmonaryvascular remodeling. This novel pathway may play an im-portant role in the early neonatal pulmonary vascular remod-eling associated with single-ventricle physiology, and thera-peutic treatment strategies targeting thrombin blockade inthese patients may have clinical utility.

Carnitine Metabolism andMitochondrial DysfunctionL-Carnitine is a trimethylated amino acid that is involved inthe transport of long-chain fatty acids across the innermitochondrial membrane (Figure 3). Carnitine is present inthe form of either free carnitine (nonesterified molecule) oracylcarnitines (esterified form). Acylcarnitines are productsof the reaction in which acyl moieties are transferred tocarnitine from acyl-CoA (acyl-coenzyme A). This reaction iscatalyzed by acyltransferases. Thus, the acylcarnitine/freecarnitine ratio is a measure of acylated carnitines versus freecarnitines. A low acylcarnitine/free carnitine ratio indicates ahealthy mitochondrion, and a high acylcarnitine/free carnitine

ratio indicates mitochondria with a reduced capacity for ATPproduction. Data in shunt lambs indicate that there is adisruption in carnitine metabolism that involves decreasedexpression of carnitine palmitoyltransferase-1 and -2, as wellas carnitine acetyltransferase. In addition, reactive nitrogenspecies appear to be involved in the disruption of carnitinehomeostasis, because nitrated carnitine acetyltransferase iselevated in shunt lambs, whereas the exposure of purifiedcarnitine acetyltransferase to authentic ONOO� decreases itsactivity.49 Together these alterations result in mitochondrialdysfunction, as demonstrated by decreased superoxide dis-mutase 2 expression, increased uncoupling protein-2 expres-sion, and an increased lactate/pyruvate ratio.49 Decreasedexpression of superoxide dismutase 2 has also been demon-strated recently in smooth muscle cells isolated from patientswith pulmonary arterial hypertension.50 It is now becomingapparent that mitochondria play a key role in the developmentof various cardiovascular diseases, including heart disease,51

diabetes mellitus,52 and atherosclerosis.53 Studies have shownthat mitochondria-targeted antioxidants have a beneficialeffect in stroke-prone rats,54 sepsis-induced cardiac dysfunc-tion,55 and cardiac ischemia-reperfusion injury,56 and there isincreasing interest in the potential of mitochondria-targetedantioxidants in the treatment of cardiovascular disease.57,58 Itremains to be seen whether this is a potential therapeuticstrategy for children with single-ventricle physiology.

Mitochondria are the major site of cellular ATP produc-tion. ATP can stimulate NO release via activation of eNOS.59

Studies have also demonstrated a key role for the 90-kDa heatshock protein (Hsp90) in the activation of eNOS,60 anddisruption of the Hsp90/eNOS complex adversely affectseNOS activation and induces eNOS uncoupling.61 Hsp90 is

Table 3. Developmental Changes in Gene Expression inShunt Lambs

Functional ClassificationNo. of GenesUpregulated

No. of GenesDownregulated

Gene functional classification in3-day shunts

Cell cycle–related genes 7 0

Cell proliferation–related genes 5 0


Extracellular matrix– and celladhesion–related genes

6 2

Transcription factors 4 0

Unclassified 90 15

Total 118 19

Gene functional classification in4-week shunts

Cell proliferation–related genes 8 0

Extracellular matrix–related genes 3 28


Specific cell signaling pathways 6 0

Transcription factors 6 0

Unclassified 132 62

Total 155 96

Figure 3. The “carnitine shuttle” and role of carnitine in themitochondrial oxidation of fatty acids. CPT-I indicates carnitinepalmitoyltransferase-1; CPT-II, carnitine palmitoyltransferase-2;CACT, carnitine acylcarnitine translocase; CrAT, carnitine acetyltransferase; Acyl CoA, acyl coenzyme A; OMM, outer mitochon-drial membrane; IMM, inner mitochondrial membrane; andCoASH, coenzyme A.





ATP dependent, and there is a temporal decrease in eNOS/Hsp90 interactions in shunt lambs that is correlated with adecline in mitochondrial function.49 Data indicate that themitochondrial dysfunction may result from an increase in thenitration of mitochondrial proteins due to an asymmetricaldimethylarginine–mediated redistribution of eNOS to themitochondria.62 Increased levels of asymmetrical dimethy-larginine have been implicated in the pathogenesis of pulmo-nary hypertension63,64 and in a single study of children andyoung adults with CHD and increased pulmonary bloodflow.65 Because studies support the view that the ratiobetween L-arginine and asymmetrical dimethylarginine is akey component in the regulation of eNOS activity, it ispossible that L-arginine could be a therapy for children withsingle-ventricle physiology.

Identification of New Genes Involved in EarlyNeonatal Pulmonary Vascular RemodelingPrevious studies have identified a burst of pulmonary angio-genesis in shunt lambs that peaks at 4 weeks of age and isthen lost; however, it is unclear how this angiogenic andapparently antiangiogenic signaling is regulated. Microarrayanalysis has been used to identify gene changes in shuntlambs at an early time point before the angiogenic burst (3days of age) and at 4 weeks of age to determine whether thereare differences in gene expression between the 2 ages andbetween the age-matched control lambs. The data indicatedthat there are a large number of gene categories that areregulated between the shunt lambs and the age-matchedcontrols at both 3 days and 4 weeks of age (Table 3).Furthermore, when only genes directly related to angiogene-

Figure 4. Relevance network of genes belonging to angiogenesis-related signaling pathways identified with Pathway Architect software.Yellow indicates the genes that are altered between shunt and control lambs at 3 days of age.





sis and the extracellular matrix are examined, the dataindicate that these genes are predominantly upregulated at 3days of age and downregulated at 4 weeks of age (Table 3).However, relevance network analyses in the 3-day-old shuntlambs reveal just how complex and interconnected thesesignaling pathways are (Figure 4). Thus, although thesestudies will likely lead to novel signaling pathways that canbe investigated to produce exciting new results, they areunlikely to lead to new therapeutic strategies in the nearfuture.

ConclusionsIn summary, with the use of an animal model that enables usto study the initial onset of neonatal pulmonary vascularremodeling associated with single-ventricle physiology, newpathways for further mechanistic studies have been identified.New potential targets for therapeutic intervention have beenidentified and are now being tested: L-Arginine, antioxidants,thrombin, and L-carnitine. In the longer term, we can hope thatthe use of newer techniques such as metabolomics, proteomics,and gene profiling may lead to new therapeutic targets to addressthe initial onset of neonatal pulmonary vascular remodelingassociated with single-ventricle physiology.

AcknowledgmentsThe authors thank the rest of the members of their respectiveresearch groups, without whom this review could not have beenwritten.

Sources of FundingThis research was generously supported by the Fondation Leducq toallow the formation of a Transatlantic Network. This work was alsofunded in part by grants HL60190 (to Dr Black), HL67841 (to DrBlack), HL084739 (to Dr Black), R21HD057406 (to Dr Black),HL61284 (to Dr Fineman), and HL08 6513 (to Dr Oishi), all from theNational Institutes of Health; an American Heart Association SoutheastAffiliates Beginning Grant-in-Aid Award (09BGIA2310050, to DrSharma); and a Seed Award from the Cardiovascular Discovery Instituteof the Medical College of Georgia to (Dr Sharma).

DisclosuresNone.

References1. Fixler DE, Nembhard WN, Salemi JL, Ethen MK, Canfield MA. Mor-

tality in first 5 years in infants with functional single ventricle born inTexas, 1996 to 2003. Circulation. 2010;121:644–650.

2. Stumper O. Hypoplastic left heart syndrome. Heart. 2010;96:231–236.3. Reddy VM, Doff BM, Phillip M, Edwin P, Frank LH. Pulmonary artery

growth after bidirectional cavopulmonary shunt: is there a cause forconcern? J Thorac Cardiovasc Surg. 1996;112:1180–1192.

4. Cleuziou J, Schreiber C, Cornelsen JK, Horer J, Eicken A, Lange R.Bidirectional cavopulmonary connection without additional pulmonaryblood flow in patients below the age of 6 months. Eur J CardiothoracSurg. 2008;34:556–562.

5. Hess J. Long-term problems after cavopulmonary anastomosis: diagnosisand management. Thorac Cardiovasc Surg. 2001;49:98–100.

6. Herget J. Animal models of pulmonary hypertension. Eur Respir Rev.1993;3:559–563.

7. Reddy VM, Meyrick B, Wong J, Khoor A, Liddicoat JR, Hanley FL,Fineman JR. In utero placement of aortopulmonary shunts: a model ofpostnatal pulmonary hypertension with increased pulmonary blood flowin lambs. Circulation. 1995;92:1–8.

8. Oishi PE, Wiseman DA, Sharma S, Kumar S, Hou Y, Datar SA, AzakieA, Johengen MJ, Harmon C, Fratz S, Fineman JR, Black SM. Progressivedysfunction of nitric oxide synthase in a lamb model of chronically

increased pulmonary blood flow: a role for oxidative stress. Am J PhysiolLung Cell Mol Physiol. 2008;295:L756–L766.

9. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthasein the lungs of patients with pulmonary hypertension. N Engl J Med.1995;333:214–221.

10. Archer SL, Djaballah K, Humbert M, Weir KE, Fartoukh M, Dall’ava-Santucci J, Mercier JC, Simonneau G, Dinh-Xuan AT. Nitric oxidedeficiency in fenfluramine- and dexfenfluramine-induced pulmonaryhypertension. Am J Respir Crit Care Med. 1998;158:1061–1067.

11. Kiettisanpipop P, Lertsapcharorn P, Chotivittayatarakorn P, PoovorawanY. Plasma levels of nitric oxide in children with congenital heart diseaseand increased pulmonary blood flow. J Med Assoc Thai. 2007;90:2053–2057.

12. Ikemoto Y, Teraguchi M, Kobayashi Y. Plasma levels of nitrate incongenital heart disease: comparison with healthy children. PediatrCardiol. 2002;23:132–136.

13. Beghetti M, Silkoff PE, Caramori M, Holtby HM, Slutsky AS, Adatia I.Decreased exhaled nitric oxide may be a marker of cardiopulmonarybypass-induced injury. Ann Thorac Surg. 1998;66:532–534.

14. Hiramatsu T, Imai Y, Takanashi Y, Hoshino S, Yashima M, Tanaka SA,Chang D, Nakazawa M. Time course of endothelin-1 and nitrate anionlevels after cardiopulmonary bypass in congenital heart defects. AnnThorac Surg. 1997;63:648–652.

15. van Beynum IM, Mooij C, Kapusta L, Heil S, den Heijer M, BlomHJ. Common 894G�T single nucleotide polymorphism in the genecoding for endothelial nitric oxide synthase (eNOS) and risk of congenitalheart defects. Clin Chem Lab Med. 2008;46:1369–1375.

16. Godfrey V, Chan SL, Cassidy A, Butler R, Choy A, Fardon T, StruthersA, Lang C. The functional consequence of the Glu298Asp polymorphismof the endothelial nitric oxide synthase gene in young healthy volunteers.Cardiovasc Drug Rev. 2007;25:280–288.

17. Veldman BA, Spiering W, Doevendans PA, Vervoort G, Kroon AA, deLeeuw PW, Smits P. The Glu298Asp polymorphism of the NOS 3 geneas a determinant of the baseline production of nitric oxide. J Hypertens.2002;20:2023–2027.

18. Bloch KD, Ichinose F, Roberts JD, Zapol WM. Inhaled NO as a thera-peutic agent. Cardiovasc Res. 2007;75:339–348.

19. Huddleston A, Knoderer C, Morris J, Ebenroth E. Sildenafil for thetreatment of pulmonary hypertension in pediatric patients. PediatrCardiol. 2009;30:871–882.

20. Tulloh R. Etiology, diagnosis, and pharmacologic treatment of pediatricpulmonary hypertension. Paediatr Drugs. 2009;11:115–128.

21. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K,Masaki T. Cloning of a cDNA encoding a non-isopeptide-selectivesubtype of the endothelin receptor. Nature. 1990;348:732–735.

22. Shetty SS, Okada T, Webb RL, DelGrande D, Lappe RW. Functionallydistinct endothelin B receptors in vascular endothelium and smoothmuscle. Biochem Biophys Res Commun. 1993;191:459–464.

23. Bradley LM, Czaja JF, Goldstein RE. Circulatory effects of endothelin innewborn piglets. Am J Physiol. 1990;259:H1613–H1617.

24. Cassin S, Kristova V, Davis T, Kadowitz P, Gause G. Tone-dependentresponses to endothelin in isolated perfused fetal sheep pulmonary cir-culation in situ. J Appl Physiol. 1991;70:1228–1234.

25. Hell JW, Yokoyama CT, Wong ST, Warner C, Snutch TP, Catterall WA.Differential phosphorylation of two size forms of the neuronal class CL-type calcium channel alpha 1 subunit. J Biol Chem. 1993;268:19451–19457.

26. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning andexpression of a cDNA encoding an endothelin receptor. Nature. 1990;348:730–732.

27. Wedgwood S, Dettman R, Black S. ET-1 stimulates pulmonary arterialsmooth muscle cell proliferation via induction of reactive oxygen species.Am J Physiol. 2001;281:L1058–L1067.

28. Giaid A, Michel RP, Stewart DJ, Sheppard M, Corrin B, Hamid Q.Expression of endothelin-1 in lungs of patients with cryptogenic fibrosingalveolitis. Lancet. 1993;341:1550–1554.

29. Fratz S, Geiger R, Kresse H, Roemer G, Hennig M, Sebening W, Hess J.Pulmonary blood pressure, not flow, is associated with net endothelin-1production in the lungs of patients with congenital heart disease andnormal pulmonary vascular resistance. J Thorac Cardiovasc Surg. 2003;126:1724–1729.

30. Gorenflo M, Gross P, Bodey A, Schmitz L, Brockmeier K, Berger F, BeinG, Lange PE. Plasma endothelin-1 in patients with left-to-right shunt. AmHeart J. 1995;130:537–542.





31. Beghetti M. Current treatment options in children with pulmonary arterialhypertension and experiences with oral bosentan. Eur J Clin Invest.2006;36:16–24.

32. Nisbet RE, Sutliff RL, Hart CM. The role of peroxisome proliferator-ac-tivated receptors in pulmonary vascular disease. PPAR Res. 2007;2007:18797.

33. Hansmann G, de Jesus Perez VA, Alastalo T-P, Alvira CM, GuignabertC, Bekker JM, Schellong S, Urashima T, Wang L, Morrell NW, Rabin-ovitch M. An antiproliferative BMP-2/PPARgamma/apoE axis in humanand murine SMCs and its role in pulmonary hypertension. J Clin Invest.2008;118:1846–1857.

34. Hansmann G, Wagner RA, Schellong S, de Jesus Perez VA, Urashima T,Wang L, Sheikh AY, Suen RS, Stewart DJ, Rabinovitch M. Pulmonaryarterial hypertension is linked to insulin resistance and reversed by per-oxisome proliferator-activated receptor-� activation. Circulation. 2007;115:1275–1284.

35. Tian J, Smith A, Nechtman J, Podolsky R, Aggarwal S, Snead C, KumarS, Elgaish M, Oishi P, Goerlach A, Fratz S, Hess J, Catravas JD, VerinAD, Fineman JR, She JX, Black SM. Effect of PPARgamma inhibition onpulmonary endothelial cell gene expression: gene profiling in pulmonaryhypertension. Physiol Genomics. 2009;40:48–60.

36. Sharma S, Kumar S, Sud N, Wiseman DA, Tian J, Rehmani I, Datar S,Oishi P, Fratz S, Venema RC, Fineman JR, Black SM. Alterations in lungarginine metabolism in lambs with pulmonary hypertension associatedwith increased pulmonary blood flow. Vascul Pharmacol. 2009;51:359–364.

37. Howell K, Costello CM, Sands M, Dooley I, McLoughlin P. L-Argininepromotes angiogenesis in the chronically hypoxic lung: a novelmechanism ameliorating pulmonary hypertension. Am J Physiol LungCell Mol Physiol. 2009;296:L1042–L1050.

38. Harrison DG, Gongora MC. Oxidative stress and hypertension. Med ClinNorth Am. 2009;93:621–635.

39. Grobe AC, Wells SM, Benavidez E, Oishi P, Azakie A, Fineman JR,Black SM. Increased oxidative stress in lambs with increased pulmonaryblood flow and pulmonary hypertension: role of NADPH oxidase andendothelial NO synthase. Am J Physiol Lung Cell Mol Physiol. 2006;290:L1069–L1077.

40. Sharma S, Grobe AC, Wiseman DA, Kumar S, Englaish M, Najwer I,Benavidez E, Oishi P, Azakie A, Fineman JR, Black SM. Lung anti-oxidant enzymes are regulated by development and increased pulmonaryblood flow. Am J Physiol Lung Cell Mol Physiol. 2007;293:L960–L971.

41. Sharma S, Kumar S, Wiseman DA, Kallarackal S, Ponnala S, Elgaish M,Tian J, Fineman JR, Black SM. Perinatal changes in superoxide gen-eration in the ovine lung: alterations associated with increased pulmonaryblood flow. Vascul Pharmacol. 2010;53:38–52.

42. Sud N, Sharma S, Wiseman DA, Harmon C, Kumar S, Venema RC,Fineman JR, Black SM. Nitric oxide and superoxide generation fromendothelial NOS: modulation by HSP90. Am J Physiol Lung Cell MolPhysiol. 2007;293:L1444–L1453.

43. Diebold I, Petry A, Djordjevic T, BelAiba RS, Fineman JR, Black SM,Schreiber C, Fratz S, Hess J, Kietzmann T, Gorlach A. Reciprocalregulation of Rac1 and PAK-1 by HIF-1�: a positive-feedback looppromoting pulmonary vascular remodeling. Antioxid Redox Signal. 2010;13:399–412.

44. Calabrese V, Cornelius C, Rizzarelli E, Owen JB, Dinkova-Kostova AT,Butterfield DA. Nitric oxide in cell survival: a Janus molecule. AntioxidRedox Signal. 2009;11:2717–2739.

45. Zickus M, Fonseca FV, Tummala M, Black SM, Ryzhov V. Identificationof the tyrosine nitration sites in human endothelial nitric oxide synthaseby liquid chromatography-mass spectrometry. Eur J Mass Spectrom.2008;14:239–247.

46. Djordjevic T, Hess J, Herkert O, Gorlach A, BelAiba RS. Rac regulatesthrombin-induced tissue factor expression in pulmonary artery smoothmuscle cells involving the nuclear factor-kappaB pathway. AntioxidRedox Signal. 2004;6:713–720.

47. Bonello S, Zahringer C, BelAiba RS, Djordjevic T, Hess J, Michiels C,Kietzmann T, Gorlach A. Reactive oxygen species activate the HIF-1�

promoter via a functional NF�B site. Arterioscler Thromb Vasc Biol.2007;27:755–761.

48. BelAiba RS, Djordjevic T, Bonello S, Artunc F, Lang F, Hess J, GorlachA. The serum- and glucocorticoid-inducible kinase Sgk-1 is involved inpulmonary vascular remodeling: role in redox-sensitive regulation oftissue factor by thrombin. Circ Res. 2006;98:828–836.

49. Sharma S, Sud N, Wiseman DA, Carter AL, Kumar S, Hou Y, Rau T,Wilham J, Harmon C, Oishi P, Fineman JR, Black SM. Altered carnitinehomeostasis is associated with decreased mitochondrial function andaltered nitric oxide signaling in lambs with pulmonary hypertension. Am JPhysiol Lung Cell Mol Physiol. 2008;294:L46–L56.

50. Archer SL, Marsboom G, Kim GH, Zhang HJ, Toth PT, Svensson EC,Dyck JR, Gomberg-Maitland M, Thebaud B, Husain AN, Cipriani N,Rehman J. Epigenetic attenuation of mitochondrial superoxide dismutase2 in pulmonary arterial hypertension: a basis for excessive cell prolif-eration and a new therapeutic target. Circulation. 2010;121:2661–2671.

51. Davidson SM. Endothelial mitochondria and heart disease. CardiovascRes. 2010;88:58–66.

52. Cheng X, Siow RC, Mann GE. Impaired redox signaling and antioxidantgene expression in endothelial cells in diabetes: a role for mitochondriaand the Nrf2-Keap1 defense pathway. Antioxid Redox Signal. 2011;14:469–487.

53. Leduc L, Levy E, Bouity-Voubou M, Delvin E. Fetal programming ofatherosclerosis: possible role of the mitochondria. Eur J Obstet GynecolReprod Biol. 2010;149:127–130.

54. Graham D, Huynh NN, Hamilton CA, Beattie E, Smith RA, CochemeHM, Murphy MP, Dominiczak AF. Mitochondria-targeted antioxidantMitoQ10 improves endothelial function and attenuates cardiac hypertro-phy. Hypertension. 2009;54:322–328.

55. Supinski GS, Murphy MP, Callahan LA. MitoQ administration preventsendotoxin-induced cardiac dysfunction. Am J Physiol Regul Integr CompPhysiol. 2009;297:R1095–R1102.

56. Adlam VJ, Harrison JC, Porteous CM, James AM, Smith RA, MurphyMP, Sammut IA. Targeting an antioxidant to mitochondria decreasescardiac ischemia-reperfusion injury. FASEB J. 2005;19:1088–1095.

57. Rocha M, Esplugues JV, Hernandez-Mijares A, Victor VM.Mitochondrial-targeted antioxidants and oxidative stress: a proteomicprospective study. Curr Pharm Des. 2009;15:3052–3062.

58. Armstrong JS. Mitochondria-directed therapeutics. Antioxid RedoxSignal. 2008;10:575–578.

59. Konduri GG, Mital S. Adenosine and ATP cause nitric oxide-dependentpulmonary vasodilation in fetal lambs. Biol Neonate. 2000;78:220–229.

60. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetro-poulos A, Sessa WC. Dynamic activation of endothelial nitric oxidesynthase by Hsp90. Nature. 1998;392:821–824.

61. Pritchard K, Stepp D, Konduri G. Inhibition of heatshock protein 90activity impairs vasorelaxation by increasing superoxide anion generationby endothelial nitric oxide synthase (eNOS). Circulation. 2001;104:500.Abstract.

62. Sud N, Wells SM, Sharma S, Wiseman DA, Wilham J, Black SM.Asymmetric dimethylarginine inhibits HSP90 activity in pulmonary ar-terial endothelial cells: role of mitochondrial dysfunction. Am J PhysiolCell Physiol. 2008;294:C1407–C1418.

63. Arrigoni FI, Vallance P, Haworth SG, Leiper JM. Metabolism of asym-metric dimethylarginines is regulated in the lung developmentally andwith pulmonary hypertension induced by hypobaric hypoxia. Circulation.2003;107:1195–1201.

64. Millatt LJ, Whitley GS, Li D, Leiper JM, Siragy HM, Carey RM,Johns RA. Evidence for dysregulation of dimethylarginine dimethyl-aminohydrolase I in chronic hypoxia-induced pulmonary hyper-tension. Circulation. 2003;108:1493–1498.

65. Gorenflo M, Zheng C, Poge A, Bettendorf M, Werle E, Fiehn W, UlmerH. Metabolites of the L-arginine-NO pathway in patients with left-to-rightshunt. Clin Lab. 2001;47:441–447.

KEY WORDS: endothelial dysfunction � endothelin � mitochondria � nitricoxide synthase � oxidative stress





Early determinants of pulmonary vascular remodeling

Health & Medicine

Transcript of Early determinants of pulmonary vascular remodeling