Archer SL

25 Pages·2010·2.88 MB·English

Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 in Pulmonary
Arterial Hypertension. A Basis for Excessive Cell Proliferation and a New
Therapeutic Target
Stephen L. Archer, Glenn Marsboom, Gene H. Kim, Hannah J. Zhang, Peter T. Toth,
Eric C. Svensson, Jason R.B. Dyck, Mardi Gomberg-Maitland, Bernard Thébaud,
Aliya N. Husain, Nicole Cipriani and Jalees Rehman
Circulation published online Jun 7, 2010;
DOI: 10.1161/CIRCULATIONAHA.109.916098
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Epigenetic Attenuation of Mitochondrial Superoxide
Dismutase 2 in Pulmonary Arterial Hypertension
A Basis for Excessive Cell Proliferation and a New Therapeutic Target
Stephen L. Archer, MD; Glenn Marsboom, PhD; Gene H. Kim, MD; Hannah J. Zhang, PhD;
Peter T. Toth, PhD; Eric C. Svensson, MD, PhD; Jason R.B. Dyck, PhD;
Mardi Gomberg-Maitland, MD; Bernard The´baud, MD; Aliya N. Husain, MD;
Nicole Cipriani, MD; Jalees Rehman, MD
Background—Excessive proliferation and impaired apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs)
contribute to vascular obstruction in patients and fawn-hooded rats (FHRs) with PA hypertension (PAH). Expression
andactivityofmitochondrialsuperoxidedismutase-2(SOD2),themajorgeneratorofH O ,isknowntobereducedin
2 2
PAH; however, the mechanism and therapeutic relevance of this are unknown.
MethodsandResults—SOD2expressioninPASMCsisdecreasedinPAHpatientsandFHRswithPAH.FHRPASMCs
havehigherproliferationandlowerapoptosisratesthanSprague-DawleyratPASMCs.Moreover,FHRPASMCshave
hyperpolarized mitochondria, low H O production, and reduced cytoplasmic and mitochondrial redox state. Admin-
2 2
istration of SOD2 small interfering RNA to normal PASMCs recapitulates the FHR PAH phenotype, hyperpolarizing
mitochondria,decreasingH O ,andinhibitingcaspaseactivity.Conversely,SOD2overexpressioninFHRPASMCsor
2 2
therapy with the SOD-mimetic metalloporphyrin Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) reverses the
hyperproliferative PAH phenotype. Importantly, SOD-mimetic therapy regresses PAH in vivo. Investigation of the
SOD2 gene revealed no mutation, suggesting a possible epigenetic dysregulation. Genomic bisulfite sequencing
demonstratesselectivehypermethylationofaCpGislandinanenhancerregionofintron2andanotherinthepromoter.
Differential methylation occurs selectively in PAs versus aortic SMCs and is reversed by the DNA methyltransferase
inhibitor5-aza-2(cid:1)-deoxycytidine,restoringbothSOD2expressionandtheratioofproliferationtoapoptosis.Expression
of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs.
Conclusions—Tissue-specific,epigeneticSOD2deficiencyinitiatesandsustainsaheritableformofPAHbyimpairingredox
signaling and creating a proliferative, apoptosis-resistant PASMC. SOD augmentation regresses experimental PAH. The
discoveryofanepigeneticcomponenttoPAHmayoffernewtherapeutictargets. (Circulation.2010;121:2661-2671.)
Key Words: epigenesis, genetic (cid:1) gene silencing (cid:1) hypoxia-inducible factor-1, (cid:1)subunit (cid:1) hypertension, pulmonary
(cid:1) mitochondrial diseases (cid:1) oxidation-reduction (cid:1) superoxide dismutase 2
Pulmonaryartery(PA)hypertension(PAH)isasyndrome musclecell(PASMC),asreviewedelsewhere.1Althoughthis
inwhichobstructed,constrictedsmallPAsandincreased study focuses on PASMCs, many cell types are abnormal in
pulmonary vascular resistance ultimately lead to right ven- PAH. There is infiltration of the lung with inflammatory
tricular hypertrophy and failure. Despite important advances cells, disorganized endothelial proliferation in plexiform
inunderstandingthemechanismofPAH,suchasthediscov-
lesions, activation of myofibroblasts, and disruption of the
ery of mutations in bone morphogenetic protein receptors in
extracellularmatrix.Theanimalmodelusedinthisstudy(the
familialPAH,andtheadventofeffectiveoraltherapies,such
fawn-hoodedrat[FHR])developsspontaneousPAH,charac-
asphosphodiesterase-5inhibitorsandendothelinantagonists,
terized by excessive proliferation and impaired apoptosis of
mortality remains high (15% at 1 year).
thePASMCs;therefore,thismodelisusefulforstudyingthe
Clinical Perspective on p 2671 roleofPASMCsinPAH.Moreover,FHRssharewithhuman
PAH may be viewed in part as a disease of excess PAH PASMCs a disrupted mitochondrial network and low
proliferation and impaired apoptosis of the PA smooth expression and activity of superoxide dismutase-2 (SOD2).2
ReceivedOctober15,2009;acceptedApril12,2010.
FromtheDepartmentsofMedicine(S.L.A.,G.M.,G.H.K.,H.J.Z.,P.T.T.,E.C.S.,M.G.-M.,J.R.)andPathology(A.N.H.,N.C.),SectionofCardiology,
UniversityofChicago,Chicago,Ill,andDepartmentsofPharmacology(J.R.B.D.)andPediatrics(B.T.),UniversityofAlberta,Edmonton,Alberta,Ontario,Canada.
Theonline-onlyDataSupplementisavailablewiththisarticleathttp://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.916098/DC1.
CorrespondencetoStephenL.Archer,MD,FAHA,FACC,FRCP(C),HaroldHinesJr.ProfessorofMedicine,ChiefofCardiology,Universityof
Chicago,5841SMarylandAve,MC6080,Chicago,[email protected]
©2010AmericanHeartAssociation,Inc.
Circulationisavailableathttp://circ.ahajournals.org DOI:10.1161/CIRCULATIONAHA.109.916098
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2662 Circulation June 22, 2010
The proliferation/apoptosis imbalance in PAH suggests 5-minuteacquisitionperiodwerequantifiedbymeasuringthepeak
similarities to cancer,3 although it should be noted that heightofelectronparamagneticresonancetripletsignal,ameasure
of3-methoxycarbonyl-proxylnitroxidekinetics.
endothelialcellsandPASMCsdonotinvadethesurrounding
tissue or metastasize. Nevertheless, FHR PAH and certain
Enhanced Chemiluminescence
cancers share a metabolic profile that creates a “pseudohy- ThePAwasincubatedinL-012(100(cid:2)mol/Lfor30minutesat37°C;
poxic” cellular environment that favors proliferation. Rele- WakoChemicals,Richmond,VA),asuperoxide-sensitivechemilu-
vant abnormalities include decreased SOD2,2,4 normoxic minescenceenhancerthatdoesnotundergoredoxcycling.2
activation of the transcription factor hypoxia-inducible
factor-1(cid:1) (HIF-1(cid:1)),2,5,6 and HIF-1(cid:1)–induced activation of Amplex Red
MeasurementofH O productionwasperformedinPASMCorPA
pyruvate dehydrogenase kinase,2,7 which inhibits the Krebs 2 2
ringswiththefluorometricAmplexRedassay.2
cycle, shifting metabolism toward glycolysis.2,7 As a conse-
quence, electron flux is reduced, and generation of reactive Immunoblotting and Immunofluorescence
oxygen species (ROS) in the mitochondria decreases. Immunoblottingwasperformedon25(cid:2)gproteinpooledfromn(cid:3)3
ThereisevidencethatimplicatesSOD2inthedevelopment fourthdivisionPAsorisolatedPASMCs(seetheonline-onlyData
of idiopathic PAH. SOD2 downregulation precedes PAH in Supplement).2
FHRs.2 In rats, the SOD2 gene is located on chromosome 1
Small Interfering RNA
and FH-BN1 consomic rats (identical to FHRs except they
PASMCs were grown to (cid:2)50% confluence and then exposed to
have introgression of a normal chromosome 1) do not
anti-SOD2 small interfering RNA (siRNA). Several “on-target”
developPAH.2Likewise,inhumanPAH,SOD2deficiencyis siRNAs(AppliedBiosystems,FosterCity,Calif)weretested,and
notedinPAsandplexiformlesions.2,4Biologicalplausibility the optimal dose to achieve maximal knockdown with minimal
for a causal role for SOD2 is supported by the recent toxicity was established. For each experiment, there was a
scrambled siRNA control. Details are given in the online-only
recognition that SOD2 is a putative tumor-suppressor gene
Data Supplement.
andthatepigeneticsilencingofSOD2increasesproliferation
of cancer cells.8,9 Replication-Deficient Adenoviruses
SOD2 is the gatekeeper that regulates physiological pro- FHRPASMCswereinfectedwithserotype5recombinantadenovi-
duction of H O (produced from mitochondrial superoxide rus carrying the human SOD2 gene under a cytomegalovirus
2 2
duringrespiration).H O islesstoxicthansuperoxide,andits promoterasdescribed2(seetheonline-onlyDataSupplement).
2 2
greater diffusion radius allows it to serve as a signaling
Redox-Sensitive Green Fluorescence
molecule.H O modulatestheactivityoftranscriptionfactors
2 2 Protein Constructs
such as HIF-1(cid:1)(which it inhibits)10,11 and sulfhydryl rich
Redox-sensitivegreenfluorescenceprotein(GFP)constructs(roGFP2)
proteins, including the voltage-gated potassium channel
targeted to either the cytoplasm or mitochondria (a kind gift of Dr
Kv1.512 (which it activates). Because FHR PAH is heritable Remington,UniversityofOregon)weretransfectedintoPASMCswith
and yet sequencing showed that the SOD2 gene has no the Fugene HD transfection reagent (Roche, Basel, Switzerland). By
mutations, we hypothesized that epigenetic suppression of usingdifferentexcitationwavelengths(400and490nm)andmeasuring
emissionat535nm,weassessedtheredoxstatusofcells(thegreater
SOD2 may initiate and/or sustain PAH. We report here that
lightemission,themoreoxidizedthePASMCs).13
selectivehypermethylationofCpGislandsintheSOD2gene
promotes a proliferative, antiapoptotic phenotype in O Consumption
2
PASMCs. Remarkably, induction of SOD2 deficiency is O consumptionwasmeasuredwithaMitocellMT200chamberand
2
sufficient to create a PAH phenotype in normal PASMCs, 782 oxygen meter (Strathkelvin Instruments, North Lanarkshire,
whereas correction of SOD2 deficiency has therapeutic ben- Scotland).Cells(106in500(cid:2)L)werestudiedinculturemediumat
37°Cduringa10-minuteprotocol.
efit in FHR PASMCs and regresses PAH in vivo. This
epigenetic mechanism may have significant mechanistic and Metalloporphyrin Mn(III)tetrakis (4-Benzoic Acid)
therapeutic implications in human PAH. Porphyrin Therapy In Vivo
Metalloporphyrin Mn(III)tetrakis (4-benzoic acid) porphyrin
Methods (MnTBAP)isasynthetic,nontoxic,cell-permeableSODmimetic.A
daily 10-mg/kg dose of MnTBAP was selected on the basis of
Animal Studies
preliminarydataandtheliterature.Similardoses(10mg/kgIPevery
The University of Chicago Animal Care Committee approved all 3days)arewelltoleratedandpreventproliferationofpremalignant
protocols. FHRs were purchased from Charles Rivers Laboratories cells in a rat model of Barrett esophagus.14 Once FHRs had
(Wilmington,Mass)andthenbredinourfacility.FHRswerestudied establishedPAH,asconfirmedbyaPAaccelerationtime(cid:4)25ms,
at (cid:2)40 weeks of age. Age-matched FH-BN1 rats and weight- on Doppler echocardiography, they were randomized to receive
matchedSprague-Dawleyrats(SDRs)servedascontrols. MnTBAP versus vehicle for 4 weeks. Assessment of functional
capacity (a graded treadmill test) and right ventricular mass and
ROS in Isolated Resistance PAs and PASMCs function(echocardiography)wasperformedbeforeandaftertherapy.
ROS were measured in isolated resistance PAs with L-012 chemi- The severity of PAH at study termination was determined by
luminescenceorelectronparamagneticresonance. Doppler echocardiography and high-fidelity catheterization (44
weeksofage).
Electron Paramagnetic Resonance
Isolated resistance PAs were incubated with a derivative of cyclic Bisulfite Sequencing
hydroxylamine(200(cid:2)mol/LinKrebs-HEPESbufferwithdesferri- Genomic DNA was isolated from resistance PAs of FH-BN1
oxamineandDETCat37°C).ChangesinROSproductionduringa consomic rats and FHRs (treated with vehicle or 5-aza-2(cid:1)-
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Archer et al SOD2 Methylation in Pulmonary Hypertension 2663
Figure1.DecreasedSOD2expressioninFHRsandpatientswithWorldHealthOrganizationCategory-1PAH.A,Confocalmicroscopy
showsdecreasedPASMCSOD2expressionandincreasedmitochondrialfragmentationinFHRPASMCs.NotethelossofSOD2
(green)inFHRsasevidencedbythereducedintensityandpeakheight.B,Confocalimmunofluorescenceimagesof3controlpatients
(whodiedofnon–lung-relatedconditions;top3rows)and3patientswhodiedofPAH(bottomrows).Notethemedialhypertrophyand
plexiformlesionsinthePAHpatients.SOD2expression(red)isdecreasedinthemediaandadventitiaofsmallPAsandintheplexi-
formlesions.(cid:1)-Smoothmuscle(SM)actin(green)isincreasedinPAH.C,Mean(cid:5)SEMofredfluorescenceintensityshowingdecreased
SOD2expressioninthemediaofsmallPAs.*P(cid:4)0.05.
deoxycytidine[5-AZA];1mg/kgbodyweightIPinjectionevery3 Detailed methods for cell culture, quantitative polymerase chain
daysfor2weeks).GenomicDNAwasdigestedwiththerestriction reaction, catheterization, echocardiography, functional capacity
enzyme HindIII and then modified with sodium bisulfite with the (treadmill), PASMC proliferation, and apoptosis assays have been
ZymoResearchEZDNAMethylationKit(ZymoResearch,Orange, publishedpreviously7andcanbefoundintheMethodssectioninthe
Calif).WedesignedprimersspecifictobisulfitemodifiedDNAin6 online-only Data Supplement. The methodology for quantitative
segments of the SOD2 promoter region (cid:2)2 kb upstream of the lunghistologyisalsofoundintheMethodssectionintheonline-only
transcriptioninitiationsiteand1segmentwithinintron2(TableIin DataSupplement.
theonline-onlyDataSupplement).Polymerasechainreactionprod- Theauthorshadfullaccesstoandtakefullresponsibilityforthe
uctswereisolatedandpurifiedbyagarosegelelectrophoresiswitha integrity of the data. All authors have read and agree to the
manuscriptaswritten.
QIAquickGelIsolationKit(Qiagen,Valencia,Calif)andclonedinto
theTOPOTAexpressionvector(Invitrogen,Carlsbad,Calif).After
Results
transformation of 1-shot, chemically competent Escherichia coli
cells,10to15cloneswereselectedforsequencing.Sequenceswere SOD2 Expression Is Reduced in PASMCs of FHRs
thenanalyzedformethylationatallCpGlocations.
and Patients With PAH
Immunostaining revealed a marked reduction in SOD2 ex-
Statistics
pressionandfragmentationofthemitochondrialreticulumin
Sample size for all experiments was (cid:3)10 per group unless stated
otherwise.Valuesarestatedasmean(cid:5)SEM.Intergroupdifferences FHR PASMCs (Figure 1A). In lung tissue harvested at
between 2 groups were assessed by an unpaired Student t test; an autopsy,SOD2expressionwasreducedwithinthemediaand
ANOVA with posthoc analysis using the Fisher protected least adventitia of small PAs and in plexiform lesions (Figure 1B
significance test was used for comparison among multiple groups. and1C)ofPAHcomparedwithcontrolpatients(withoutlung
Normality was confirmed with a Kolmogorov-Smirnov or disease).
D’Agostino and Pearson omnibus test. For data that were not
normallydistributed(PAmuscularizationaftercontrolorMnTBAP
SOD2 Knockdown Recapitulates a “PAH
treatment), we used a Mann-Whitney test. A Fisher exact test was
Phenotype” in Normal PASMCs
used to compare the number of muscularized vessels between
MnTBAPanduntreatedFHRs.AvalueofP(cid:4)0.05wasconsidered To assess whether downregulation of mitochondrial SOD2
statisticallysignificant. wassufficienttocontributetotheinitiationofPAH,weused
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2664 Circulation June 22, 2010
Figure2.InhibitionofSOD2expressionin
normalSDRPASMCrecapitulatesthe
PAHphenotype.A,siRNAsreduceSOD2
mRNAandH O production.B,siSOD2
2 2
hyperpolarizes(cid:6)(cid:4)m(increasedredTMRM
intensity).C,SOD2siRNAactivates
HIF-1(cid:1)(notetranslocationtothenucleus;
redinmiddle)anddecreasesKv1.5
expression(decreased;greeninleft).D,
ImmunoblottingconfirmedthatsiSOD2
decreasesKv1.5expression(left).Inline
withlossofKvchannels,siSOD2
increasedintracellularK(cid:7)concentration
(middle).siSOD2alsoreducedcaspase
activity(right).TMRMindicatestetrameth-
ylrhodaminemethyl-esterperchlorate;
aFU,arbitraryfluorescentunits.
SOD2 siRNA to reduce SOD2 levels in normal SDR gene and examined the effects of demethylation using the
PASMCs. SOD2 siRNA decreased SOD2 expression (mes- methyltransferase inhibitor 5-AZA. Genomic DNA isolated
senger RNA [mRNA] and protein; Figure 2 and Figure I in from PAs of FHRs, consomic FH-BN1 rats, and 5-AZA–
the online-only Data Supplement). Small interfering SOD2 treated FHRs was analyzed. Although there are many CpG
also decreased H O production (Figure 2A). SOD2 knock- islands in the SOD2 gene, differential hypermethylation
2 2
down reduced the production of superoxide in normal (relative to consomic FH-BN1 DNA) was found only in an
PASMCs,asdemonstratedbyareductioninL-012chemilu- enhancer region in intron 2 (Figure 3A and 3B). To test the
minescence. The L-012 signal is specifically inhibited by effectsof5-AZAinvivo,wetreatedFHRsandthenisolated
pegylatedSODbutnotpegylatedcatalase,confirmingthatit genomic DNA from PAs for SOD2 gene methylation analy-
isameasureofsuperoxide(FigureIIintheonline-onlyData sis. 5-AZA decreased methylation of an intron 2 sequence
Supplement). The mitochondrial membrane potential ((cid:6)(cid:4)m) (Figure3Aand3B).WenextculturedPASMCsfromFHRs
ofSOD2siRNA-treatedcellsbecamehyperpolarized(Figure andSDRsandtreatedthemwith5-AZA.Again,weidentified
2B).WeinvestigatedtheeffectsofSOD2siRNAon2known adifferentiallymethylatedregionwithinintron2(Figure3C),
abnormalities implicated in creating the excess ratio of which was demethylated in response to 5-AZA. There was
proliferation to apoptosis in FHR PAH: HIF-1(cid:1)activation tissue heterogeneity in the differentially methylated sites,
(translocation to the nucleus) and Kv1.5 downregulation.2 with methylation being present in the SOD2 gene isolated
SOD2 siRNA activated HIF-1(cid:1) and downregulated Kv1.5 from FHR PA but not in FHR aortas. An additional site of
channel protein expression (Figure 2C and 2D) in SDR differential methylation was found in the FHR SOD2 pro-
PASMCs.ThelossoftheKv1.5channelhad2consequences moter (Figure III in the online-only Data Supplement).
associated with apoptosis resistance and increased cell pro- 5-AZA increased SOD2 mRNA (Figure 3D) and protein
liferation,7,15namelyincreasedcytosolicK(cid:7)(Figure2D)and (Figure 4A and 4B) in FHR PASMCs in a dose-dependent
Ca2(cid:7) (Figure I in the online-only Data Supplement). This manner (maximum, 80% SOD2 induction at 10 (cid:2)mol/L).
elevation of cytosolic K(cid:7) was associated with decreased 5-AZA also restored Kv1.5 expression (Figure 4C).
caspaseactivityinPASMCs(Figure2D).7Therefore,reduc-
ing SOD2 expression recapitulated many abnormalities seen SOD2 Hypermethylation Promotes a Proliferative,
in FHR PASMCs. Apoptosis-Resistant Phenotype
Using flow cytometry, we confirmed that under identical
Hypermethylation of the SOD2 Gene in FHRs and conditions FHR PASMCs had roughly double the rate of
the Therapeutic Effects of the Methyltransferase proliferationandhalftherateofapoptosisasSDRPASMCs
Inhibitor 5-AZA (Figure 4D and 4E). Remarkably, incubating FHR PASMCs
Sequencing of the entire SOD2 gene, beginning 600 bp andlungadenocarcinomaA549cellswith5-AZAfor3days
upstream from the transcriptional start site in SDRs and significantly decreased proliferation and caused a modest
FHRs, demonstrated identical sequences (data not shown). increase in apoptosis (Figure 4D and 4E). O consumption
2
This suggested that decreased SOD2 expression in FHRs increased in FHR PASMCs in response to 5-AZA (Figure
might reflect epigenetic silencing of gene transcription, as 4F). These data suggest that methylation-induced SOD2
occurs in cancer.16,17 We validated a bisulfite sequencing downregulation creates a low-O -consumption metabolic
2
assay to measure methylation of key regions of the SOD2 state that favors proliferation and suppresses apoptosis.
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Archer et al SOD2 Methylation in Pulmonary Hypertension 2665
Figure3.MethylationofSOD2inFHRPASMCisreversibleby5-AZA.A,SchematicoftheCGdinucleotidepercentageandCpGislands
withintheSOD2promoterandfirst2kbafterthetranscriptionalstartsite.SevenampliconsweresurveyedwithintheSOD2gene.Their
approximatelocationsarerepresentedbysolidhorizontallines.ThepositionsofindividualCpGdinucleotidesareshownasverticaltick
marksbelowtheampliconmap.NomethylatedCpGpairswereidentifiedinamplicons3to6.*DifferentiallymethylatedCpGdinucleotidesin
FHRswithinamplicon7vsBN1tissue.B,CorrespondingmethylationpercentageofthedifferentiallymethylatedCpGinintron2.Resultsare
expressedasafrequencyofcytosinemethylationinPAsfromconsomiccontrolBN1rats(n(cid:3)2),FHRs(n(cid:3)3),andFHRstreatedwith5-AZA
(FHR-Tx;n(cid:3)3).C,RepresentativesequencingtracesofgenomicDNAfromculturedPASMCs.Onlymethylatedcytidinesareprotected
againstbisulfite-mediateddeaminationofcytidineintouridine(whichisrecognizedasthymidinewhenthepolymerasechainreactionproduct
isamplified).Asindicatedbythearrow,thecytidineinFHRPASMCswasmethylated(andthereforeremainsacytidine;topleft);thisis
reversedby5-AZA.ThesiteisnotmethylatedinFHRaorticSMCsorinSDRPASMCs.Thebargraphshowsthemeandataindicatingthe
reversibilityandtissuespecificityofthisSOD2methylationinintron2inculturedPASMCs.D,FHRPASMCshavelowerSOD2mRNAlevels
vsconsomicPASMCs.5-AZAcausesadose-dependentincreaseinSOD2expression.
FHR PASMCs Produce Less ROS and Are in a Supplement). Thus, there is a reversible, methylation-induced
Reduced State depressionofH O productioninFHRs.
2 2
Baseline L-012 chemiluminescence was lower in FHRs com-
Increased Expression of DNA Methyltransferases
paredwithcontrolPASMCs(Figure5A),andthiswasmirrored
in FHRs
inareducedredoxstateinthecytoplasmandmitochondria,as
BecausegenemethylationisdependentontheactivityofDNA
measured with compartment-specific roGFPs (Figure 5B and
methyltransferases (MTs), we measured their expression in
FigureIVintheonline-onlyDataSupplement).Consistentwith
FHRs and control rats. In lung tissue, both the maintenance
this, SOD2 siRNA reduced L-012 chemiluminescence produc-
DNA MT1 and the de novo DNA MT3B were significantly
tion in PASMCs (Figure IIA in the online-only Data Supple-
upregulatedcomparedwithcontrol(Figure6A);asimilarpattern
ment) and 5-AZA restored ROS production in FHR PAs, as
wasfoundinPASMCsfromFHRs(Figure6B).
assessedbybothchemiluminescenceandelectronparamagnetic
resonance (Figure 5C). Conversely, 5-AZA had no effect on SOD2 Augmentation Restores FHR PASMC
H O production in normal PAs. Control experiments with Gene Expression
2 2
pegylatedSODandpegylatedcatalasedemonstratethespecific- Because 5-AZA is a nonspecific demethylating agent and
ity of our measurements (Figure IIB in the online-only Data may affect a multitude of genes, we investigated the thera-
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2666 Circulation June 22, 2010
Figure4.5-AZArestoresSOD2expression,reducesproliferation,andenhancesapoptosisinFHRPASMCs.AandB,Representative
andmeandatashowingthatFHRPASMCsexpresslessSOD2thanSDRPASMCs;thisispartiallyreversiblewith5-AZAtreatment.
n(cid:3)4pergroup.*P(cid:4)0.05.C,5-AZA–induced,dose-dependentincreaseinKv1.5mRNAinFHRPASMCs.*P(cid:4)0.05.DandE,5-AZA
(blackbars)increasesapoptosisanddecreasesproliferationinFHRPASMCsandlungcancercells(A549)butnotSDRPASMCs.n(cid:3)4
to6pergroup.*P(cid:4)0.05betweenbaselineand5-AZA.†SignificantdifferencefromSDRPASMCs.F,5-AZAincreasesO consumption
2
inFHRandSDRPASMCsbutnotA549cells(n(cid:3)3to8pergroup).
peuticeffectsofdirectSOD2augmentationtoassessitsrole Adenoviral SOD2 significantly increased SOD2 expres-
in PAH. We used 2 complementary strategies: direct SOD2 sion in FHR PASMCs (Figure 7A). This inactivated the
augmentation with adenoviral gene transfer carrying mito- spontaneously active HIF-1(cid:1) seen in FHRs and increased
chondrially targeted SOD2 and administration of a Kv1.5 expression (Figure 7B). Likewise, incubation of FHR
membrane-permeable SOD analog, MnTBAP. PASMCs with MnTBAP for 48 hours inactivated nuclear
Figure5.LowerROSlevelsandreduced
cellularenvironmentinFHRPASMCs.A,
DecreasedROSlevelsinfreshlyisolated
resistancePAsfromFHRsvsconsomic
ratsmeasuredwithL-012chemilumines-
cenceat37°C.B,PASMCsweretrans-
fectedwithredox-sensitiveGFPconstructs
targetedtothecytoplasm(top)andmito-
chondria(bottom).FHRPASMCshad
reducedcytoplasmandmitochondriarela-
tivetoSDRPASMCs.Dithiothreitoland
tert-butylhydroperoxidewereusedtocom-
pletelyreduceandoxidizethecells,respec-
tively.C,ROSmeandataandrepresenta-
tivetrace(inset)showingthecyclic
hydroxylamine(CMH)tripletsignal.Peak
height(electronparamagneticresonance
[EPR]amplitudeunits)isproportionalto
ROSlevelsandisnormalizedtowet
weight.PAsfromFHRstreatedwith5-AZA
(blackbars)haveincreasedproductionof
ROS(left)andH O (right)vsuntreated
2 2
FHRs.Controlratsshownoinductionof
ROSorH O ,n(cid:3)5pergroup.
2 2
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Archer et al SOD2 Methylation in Pulmonary Hypertension 2667
PAH. This may be relevant for clinical PAH because SOD2
is also downregulated in human PAH (Figure 1). We identi-
fiedhypermethylationofaCpGislandinanenhancerregion
within intron 2 and the promoter of SOD2 as the basis for
SOD2 downregulation in FHRs. This appears to reflect
significantly higher expression of DNA MT1 and MT3B in
lungs and of DNA MT3B in isolated PASMCs of FHRs
(Figure6).ThisepigeneticdownregulationofSOD2impairs
H O -mediated redox signaling, activates HIF-1(cid:1), and cre-
Figure6.DNAMTexpressionisincreasedinFHRlungand 2 2
PASMCs.A,DNAMT1and3BmRNAareincreasedinFHRvs atesaproliferative,apoptosis-resistantstate(Figures4,5,and
SDRlungs(n(cid:3)12each).*P(cid:4)0.05,**P(cid:3)0.01.B,Inlow-passage 7). Both the mechanism of SOD2 downregulation and its
(3to4)PASMCs(n(cid:3)8ineachgroup),FHRshadhigherDNA
consequences parallel those recently discovered in human
MT3BexpressionandatrendtowardincreasedDNAMT1.
cancer,16,17 but to the best of our knowledge, this is the first
reportofanepigeneticcauseofapulmonaryvasculardisease.
HIF-1(cid:1)(Figure 7C and 7D) and restored Kv1.5 expression
The epigenetic downregulation of SOD2 was reversible by
(Figure 7D).
treatment with the DNA MT inhibitor 5-AZA. Furthermore,
our study demonstrates that augmentation of SOD2 (by 3
SOD2 Augmentation Regresses PAH In Vivo
complementary strategies) restores mitochondrial function,
TotestthetherapeuticeffectsofSOD2augmentationinvivo,
inhibitsPASMCproliferation,andincreasescellapoptosisin
wetreatedFHRswithprovenPAHwithMnTBAP.MnTBAP
vitro. In vivo, MnTBAP causes partial regression of estab-
waschosenoverSOD2becauseoftheverytransientupregu-
lationoftransgeneexpression((cid:2)2weeks)thatisoftenfound lishedPAHanddecreasesthemuscularizationofpulmonary
precapillary resistance vessels (Figure 8A and 8D).
with adenoviral gene therapy. MnTBAP doubled PA accel-
The FHR SOD2 deficiency is the consequence of the
erationtime(Figure8A)andcausedaconcomitantreduction
covalent cytosine methylation that occurs in dinucleotide
inthemeanPApressure(Figure8B)andrightventricularfree
wall thickness (Figure 8A). During the 4-week treatment, CpGs (Figure 3). Methylation at the C5 atom of cytosine
exercise capacity deteriorated in vehicle-treated FHRs, inhibits gene expression by preventing the binding of tran-
whereas the MnTBAP group showed improvement (Figure scriptionfactors.18Methylationisreversibleandheritableand
8C). Quantitative histology performed by blinded readers can be tissue specific.19 Indeed, SOD2 methylation occurred
demonstratedthatMnTBAPcausedasignificantreductionin in the FHR PA but not in the FHR aorta (Figure 3), thus
the percent medial thickness of precapillary resistance arter- pointingtotissue-specificepigeneticmechanisms.Thisfind-
iesandanincreaseinthepercentageofsmall(25to50(cid:2)m) ingmaycontributetoourunderstandingofthelocalizationof
nonmuscular PAs (Figure 8D). the pathology in human and FHR PAH to the pulmonary
circulation. The SOD2 promoter and several of its introns
Discussion haveCpGislandsthatofferpotentialmethylationsites.Using
Oneofthemajorfindingsofthisstudyistheidentificationof genomic bisulfite sequencing, we directly confirmed that
SOD2methylationasapotentialnew,epigeneticmechanism FHRshave2discretesitesofdifferentialhypermethylation(1
for PAH in an animal model of spontaneous and heritable in intron 2, 1 in the promoter; Figure 3A through 3C and
Figure7.SOD2supplementationinacti-
vatesHIF-1(cid:1)andincreasesKv1.5expres-
sioninFHRPASMCs.A,TheSOD2virus
andacontrolGFPvirusareeffective,
increasingexpressionofSOD2andGFP
mRNA,respectively,inFHRPASMCs.B,
ImmunofluorescencewithanAlexa647
secondaryantibodyshowsthattheSOD2
adenovirus(Ad-)increaseshumanSOD2
expression(green;bottomleft).This
restoresKv1.5expression(green)andin-
activatesHIF-1(cid:1)(rednuclei).Nucleiare
counterstainedblue(DAPI).CandD,A
48-hourincubationwithMnTBAP
decreasesnuclearlocalizationofHIF-1(cid:1)
andrestoresKv1.5expression.
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2668 Circulation June 22, 2010
Figure8.MnTBAPregressesPAHinFHRs.A,MnTBAPreducesmeanPApressuremeasuredbyDoppler(lengthensPAacceleration
time[PAAT])anddecreasesrightventricular(RV)thicknessinFHRstreatedfor4weeks(n(cid:3)5pergroup).*P(cid:4)0.05.B,MnTBAPtherapy
reducesmeanpulmonaryarterypressure(PAP)andtotalpulmonaryresistance(TPR).C,FHRstreatedwithMnTBAPexerciselongeron
agradedtreadmill(n(cid:3)15pergroup).D,LungsectionswerestainedforvonWillebrandfactor(vWF;red),(cid:1)-smoothmusclecell(SMC)
actin(green),andDAPI(blue).Notethefullymuscularized(whitearrows),partiallymuscularized(yellowarrows),andnonmuscularized
bloodvessels(redarrows).Bottom,ArepresentativefullymuscularizedPAinavehicle-treatedFHR(left)vsMnTBAP(right).Theper-
centmedialthicknessofprecapillaryresistancePAswasreducedandthenumberofnonmuscularizedresistancePAswasincreased
byMnTBAP.**P(cid:4)0.01vscontrol.
Figure III in the online-only Data Supplement). Although Itisinterestingthatchangesintheactivityorexpressionof
manyregionsintheSOD2geneareheavilymethylated,these DNAMTscanhavearelativelyspecificoutcomeintermsof
2 were the only CpG islands that were both differentially site-specific methylation and regulation of specific genes in
methylatedanddemethylatedby5-AZA.Theintron2siteis certaintissues.Currentmodelssuggestthatthespecificityof
inthesameregionoftheSOD2genethathasbeenidentified DNA MT activity can depend on their expression levels or
in transformed human lung fibroblast cell lines.20 5-AZA their interaction with other epigenetic regulators. In the
covalentlybindsandirreversiblyinhibitsDNAMTs,21result- presentstudy,forexample,wefoundthatFHRanimalswith
ing in reactivation of transcription of previously methylated
PAHhadhigherexpressionlevelsofDNAMT3B.ThisDNA
genesafterarequisitecelldivision.Itisunclearwhy5-AZA
MThasbeenshowntohavedistinctCpGmethylationactivity
hadnoeffectonotherareasofmethylation,butotherstudies
patterns that depend on the tissue environment and on the
have described similar observations.17
expressionofdifferentiallysplicedisoforms.24Anotherlevel
CpGmethylationisestablishedandmaintainedby3DNA
ofDNAmethylationspecificityisachievedbytheinteraction
MTs.22 DNA MT1 is expressed in proliferating tissues, and
ofDNAMTswithotherepigeneticregulatorssuchashistone
its activity is coupled to DNA replication, copying methyl-
deacetylases. For example, DNA MT3B contains an ATRX
ation patterns from the parental to the daughter strand.
homologydomainthatinteractswithhistonedeacetylase1.25
Therefore, it is considered primarily a maintenance MT. In
Interestingly, such an association between increased CpG
contrast, DNA MT3A and MT3B are considered de novo
methylation and decreased histone acetylation has been ob-
MTsthatcanestablishnewmethylationpatterns.Weidenti-
fied an upregulation of DNA MT3B in FHR PASMCs served for the SOD2 gene.16
(Figure 6), which would be an elegant explanation for the Gene methylation has an established role in promoting
observed hypermethylation of the SOD2 gene. Interestingly, pathological cell proliferation in cancer. SOD2, a candidate
DNAMT3Bdepletionissufficienttoreactivatemethylation- tumor-suppressor gene,8,9 is silenced in several malignan-
silenced genes and to decrease proliferation in both human cies.16,17,26 In multiple myeloma and pancreatic carcinoma,
breast adenocarcinoma and A549 cells.23 the epigenetic silencing of SOD2 is caused by hypermethyl-
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Archer et al SOD2 Methylation in Pulmonary Hypertension 2669
ation of CpG islands within the promoter for SOD2.17,26,27 Therapeutic Implications
Demethylation of SOD2 in cancer restores SOD2, increases Evidence for the relevance of SOD2 deficiency in PAH
H O ,anddecreasescellproliferationandtumorgrowth,16,17,26 comes from the concordant benefit of 3 different strategies
2 2
consistent with our observations (Figures 3D, 4D, and 4E). thatrestoreSOD2expressionorSODactivityinourstudy.In
The effects of directly overexpressing SOD2 (by 3 comple- FHRPASMCs,SOD2genetherapy,applicationoftheSOD2
mentary means) are concordant with the effects of SOD2 mimeticMnTBAP,or5-AZAeachledtoHIF-1(cid:1)inactivation
demethylation in cancer28 (Figure 7A and 7B). Our study and restoration of Kv1.5 expression (Figure 7). Although
demonstratesthatreversinggenemethylationisbeneficialin thesesurrogateendpointsareimportant,thekeytherapeutic
FHR PASMCs in vitro. Treatment with 5-AZA decreases finding of our study is that MnTBAP treatment regresses
SOD2 methylation and causes a dose-dependent increase in PAHinvivo(Figure8Aand8D).Thishemodynamicbenefit
SOD2 and Kv1.5 expression in FHR PASMCs (Figure 4A isassociatedwithareductioninrightventricularhypertrophy
through 4C). and improved functional capacity and lung histology. The
Production of H O is a critical link between SOD2 concordantfindingsinresponsetocomplementarystrategies
2 2
expression and regulation of proliferation (Figure V in the to modulate SOD2 expression reduce the possibility of
online-only Data Supplement provides a schematic repre- artifacts related to flaws in any single strategy such as the
sentation of the cascade of the proposed transcriptional, stress of intraperitoneal MnTBAP injections, inflammation
metabolic, and redox consequences of SOD2 downregula- fromtheSOD2adenovirus,andpotentialconfoundingeffects
tion). Simply knocking down SOD2 expression in a normal of demethylation of nontarget genes by 5-AZA.
The benefits of MnTBAP are consistent with those of
PASMC diminishes endogenous H O production and leads
2 2
to an associated activation of HIF-1(cid:1)(Figure 2). Moreover, tempol (another membrane permeable SOD mimetic),
ananti-SOD2siRNAcausedmanyoftheotherabnormalities whichdecreaseshypoxicpulmonaryhypertensioninrats,31
and recombinant SOD1, which ameliorates persistent pul-
seen in human and FHR PAH, including a decrease in
monary hypertension in newborn lambs.32 Bowers et al4
expressionofKv1.5channelsandariseincytosoliccalcium
have also noted decreased SOD2 in the PAs in human
(FigureIBintheonline-onlyDataSupplement).Conversely,
PAH; however, their interpretation was that PAH is a
augmentingSOD2elevatesproductionofH O inFHRPAs
2 2 condition of increased oxidative stress (in part because
(Figure 5C) and reduces cell proliferation (Figure 4E). Sim-
other oxidant markers were elevated). Our data indicate
ilarly, in prostate cancer, overexpressing SOD2 increases
that in FHRs, H O is subphysiological because of both a
H O andreducescellproliferation.29Thus,inbothPAHand 2 2
2 2 primary effect, methylation-induced reduction of the
cancer,thereisaninversecorrelationbetweenSOD2activity
SOD2 gene expression, and a secondary effect, pyruvate
and H O -mediated cell proliferation.29 Catalase, which re-
2 2 dehydrogenase kinase–mediated inhibition of oxidative
duces H O levels, prevents the ability of SOD2 to inhibit
2 2 metabolism.2Identificationofthetherapeuticpotentialfor
cancer cell proliferation.29 These observations suggest that
5-AZAisparticularlyrelevantbecause5-AZA(decitabine)
the effect of SOD2 augmentation in PAH and cancer is
is approved for human use in myeloproliferative disorders
mediated by increasing H O production (from a deficient
2 2 (where it demethylates p15).
starting level). We support this contention by showing that
5-AZA increases H O production only in FHR PASMCs,
2 2 Limitations
having little effect on normal PASMCs (Figure 5C).
There are limitations in using human tissue. One notable
Our study also identifies the downstream mechanism by
limitation is that we are unsure about smoking status. We
whichthismitochondrialabnormalitypromotescellprolifer-
acknowledge that smoking could change SOD2 levels33;
ation, namely normoxic activation of HIF-1(cid:1). HIF-1(cid:1)acti-
however, this is unlikely to be an important confounder
vation has previously been identified in human PAH2,6 and because the rodent data are consistent with the human data.
FHR PAH.2 The present study supports a key role for H2O2 We acknowledge that the net effect of loss of SOD2 is
as the link between SOD2 and cell proliferation, as schema- controversial (oxidation in SOD2 knockout mice34 versus
tized in Figure V in the online-only Data Supplement. reduction in our study). Interestingly, the SOD2 haploinsuf-
Although ROS are toxic at high levels, there is a physiolog- ficientmousehasadoublingofcancerrisk,34supportingour
icallevelofH2O2productionbymitochondriaduringnormal central finding that downregulation of this mitochondrial
oxidativemetabolism.12,30PhysiologicallevelsofH2O2serve enzymeisaproliferative,antiapoptoticsignal.Wesuspectthe
as redox signaling molecules, involved in oxygen sensing.12 differences in ROS relate to the lifelong duration and more
The evidence linking SOD2 levels to ROS in PAH is clear. profound severity of SOD2 loss for the knockout mouse
Simply lowering SOD2 message levels (with siRNA) de- compared with acquired and more modest SOD2 loss in
creases mitochondrial H O production (Figure 2A); con- FHRs.
2 2
versely,demethylatingtheSOD2geneenhancerinFHRPAs Finally,pyruvatedehydrogenasekinaseactivationinPAH
restores SOD2 expression, leading to increased ROS and thwartsmitochondrialrespiration,limitinginputtothemito-
mitochondrial H O production (Figure 5C). Compartment- chondrialelectrontransportchain.AlthoughinFHRslowROS
2 2
specific, redox-sensitive GFPs demonstrated that the net andareducedstateactivateHIF-1(cid:1),othersfindthathighROS
change in redox state in FHR (versus SDR) PASMCs is levelsstabilizeHIF-1(cid:1).35–37Interestingly,Kaewpilaetal37noted
reduction (in both the cytosol and mitochondria), consistent in cancer that siSOD2 activates HIF-1(cid:1), consistent with our
withtheobserveddecreasedROSlevelsinFHRs(Figure5). findings.However,theyfoundthatsiSOD2increasedsuper-
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