Expression profiling
of
cardiovascular
disease
Stephen
Archacki and Qing Wang*
CenterforMolecularGenetics, Department of MolecularCardiology, LernerResearch Institute;CenterforCardiovascularGenetics, Department of CardiovascularMedicine, The Cleveland Clinic Foundation, Cleveland, OH, USA;and Department of Biological, Geologicaland EnvironmentalSciencesCleveland State University, Cleveland, OH 44115, USA
*Correspondenceto: Tel:þ1 2164450570;Fax:þ1 2164442682;E-mail:wangq2@ccf.org
Datereceived(in revised form):30th April 2004
Abstract
Cardiovasculardisease is themostimportantcauseofmorbidityandmortalityindeveloped countries, causingtwice as manydeathsas
cancerin the USA.Themajorcardiovasculardiseases, including coronaryarterydisease(CAD),myocardialinfarction (MI), congestive heart
failure(CHF)and commoncongenitalheartdisease (CHD), are caused by multiple genetic and environmentalfactors, as wellas the interactionsbetween them.Theunderlyingmolecular pathogenicmechanismsfor these disordersarestill largely unknown, butgene expression may playa central role in the developmentandprogression of cardiovasculardisease.Microarraysare high-throughputgenomic
tools thatallow the comparison of globalexpressionchangesin thousands of genesbetween normaland diseased cells/tissues.Microarrays
haverecentlybeenappliedtoCAD/MI, CHF and CHDto profile changesingene expression patternsindiseased andnon-diseasedpatients.
This sametechnologyhasalsobeen usedtocharacterise endothelialcells,vascular smoothmuscle cellsand inflammatorycells,withor without various treatments that mimic diseaseprocessesinvolved inCAD/MI.Thesestudieshaveledto the identification ofuniquesubsets of genesassociatedwithspecific diseasesand diseaseprocesses.Ongoingmicroarray studiesin the fieldwill provide insightsinto the
molecular mechanism of cardiovasculardisease andmaygeneratenewdiagnostic andtherapeuticmarkers.
Keywords:microarrayanalysis, expression profiling, coronaryarterydisease, heartfailure, congenitalheartdisease, endothelialcells,smoothmuscle cells, inflammation
I
ntro
d
u
c
t
i
on
Gene expressionis thought tobe central to thepathogenesis or progression of coronaryartery disease(CAD)/atherosclerosis, congestive heartfailure(CHF)and common congenitalheart disease(CHD).Microarrayanalysisisapowerful technique for high-throughput, global transcriptonomicprofilingof gene expression.It holdsgreat promise foranalysingthe genomic basis of variouscomplexdiseasesand permits the analysis of thousands of genes simultaneously, both indiseased and non-diseasedtissuesand/orcell lines.
Microarraysaremade bydepositingspots of DNAona solidsupport such asa coated glass surface.1A flatglass surface
makesit possible(i) toarray moleculesinaparallelfashion, (ii) to miniaturisethe procedure and (iii) to use fluorescent dyesfordetectionand thus avoidradioactivity. Thisdiffersin several waysfromconventional methods such asfilter-based supports of charged nylonandnitrocellulose for studying mRNA expression.
Avariety of technologieshasbeen used to produce microarrays.Spotted cDNA arrays,produced bydeposition of
polymerase chain reaction (PCR) products, and GeneChip oligonucleotide arrays (Affymetrix; Santa Clara, CA), pro-duced byin situsynthesis ofoligonucleotides, have both been usedsuccessfully.Microarrayscan nowbetailoredtofocus on aspecificset of genes (egthe Cardiochip,with genes related to the cardiovascular system only).The advantages of cDNA arraysarethat theyare focusedtoaspecificpathway or functionalclass of genesand willidentify the gene(s) of particular interestin the tissuestudied. Useofthis smaller chip is lessexpensive, andthesamesample canbeusedto comparesimilargenesin several species. Thelimitation of cDNA arrays, however, is that onlyavery limitedset of genes canbe evaluated. Oligonucleotide arrayshavethe advantage of allowingoneto monitoreverygene in a genome at the sametime, rather thanfocussingonaparticular subset of genes. The disadvantage is that this method isexpensive and the arrays mustbemade commercially.At this time,onlya few organisms (human,mouse and rat)are available for studies.
Microarrayscan servetocomplement othergenetic and genomictools, includingpositionalcloning2andproteomics,3
orfacilitatethe development of CAD/atherosclerosis, CHF orcommonCHD.In this paper,the authors review the current status ofmicroarray studiesin profiling gene
expressionincardiovasculardisease,with aparticularfocus on human tissuesand cells.
E
xpr
e
ss
i
on pro
fi
l
i
n
g
o
f CAD
CAD iscaused byanaccumulation of atheroscleroticplaques (atherosclerosis)in thewalls ofthe coronaryarteries. Athero-sclerosis leads to symptoms of stable angina ifnoclinically apparent plaqueruptureor significantbreachofthe arterial wall occurs, but it will resultin unstable angina, acute myo-cardialinfarction (MI) or suddencardiac deathoncethe arterial wall integrity is lostand thrombosishas occurred.
In the USA andmost otherWesterncountries, CAD is the leading causeofmorbidityandmortality.In the USA alone, it caused almost onemilliondeathsin 1999—twicethenumber caused bycancerand ten times the numbercaused by acci-dents.4 Despitesignificant medicaladvances, MI duetoCAD
(and strokes)is responsible for more deaths thanall other causescombined.
There aretwo main theories that explain the development of atherosclerosis.First, highlevels of cholesterolin the blood injurethe endothelialcells of a coronaryartery, causing an inflammatory reactionand enabling cholesterolandotherfatty materials toaccumulatethere.Secondly,repeated injury to the wall of the artery may occur throughvarious mechanisms involvingthe immunesystem orbydirect toxicity with sub-stances such as nicotineorhomocysteine,leading to the recruitmentand accumulation of inflammatory cells.5Inboth
cases, there are changes thatcan lead to the formation of atheromasand plaques.Thesetwo theories probably overlap and arenot mutuallyexclusive.Todate,several microarray studieshave been reportedto profile gene expressioninCAD using human tissues; onestudyfrom the authors’group focusedon the identification of genesdifferentiallyexpressed
inCAD and non-CAD coronary arteries,6 and theother
studieshave characterised gene expressioninatherosclerotic plaquesand plaquerupture.7–10
Four microarray studieshaveso farbeen reported for the characterisation of atherosclerosis,oneof which focusedon restenosisaftercoronaryinterventions.9The currentauthors
usedoligonucleotide arrays to study.12,000 genesfor their expressioninhumancoronaryarteries,thetarget organ of CAD(Table1).6 Fifty-six genes showed differential
expression.In the atherosclerotic coronaryartery tissues,the expression of 55 genes wasincreased,whereas only one gene — encoding glutathione-S-transferase(GST), areducing agent —showed downregulated expression. This study detectedthe expression of genes previously linkedto the generation of CAD,such asosteopontinexpressed in smooth muscle cells,vascularcelladhesion molecule(VCAM)-1expressed inendothelialcellsand matrix metalloprotease (MMP)-9 expressed in macrophages.This lendscredibility toexpression profiling asavalid approach for studying gene expressionin CAD.
The associations of 49genesappearedtobenoveland had not previouslybeen shown tobe associatedwith CAD.These included genesencoding retinoic acid responderbinding (RAR) protein, butyrophilin, steroidogenic acuteregulatory protein (STAR),provirusintegration site forMoloney murine leukemiavirus-2 (PIM-2),signal transducerand activator of transcription-91 (STAT-91) and cathepsins K and H.The RARgene is regulated by vitaminAsignalling pathwaysand upregulates the expression ofthescavenger receptorCD36;11
butyrophilinhas receptorfunctions that mediatethetransfer of lipid;12and cathepsinsK and H arelysosomal proteases
involved in proteindegradation.13,14STAR is therate-limiting
step in steroidogenesisfor thetransfer of cholesterolinto the mitochondria.15 ThePIM-2gene is presentin mitogenically
stimulated hematopoietic cells16and isexpressedwidelyin
themediaof coronaryarteries.6TheSTAT-91gene isasignal
transducerand activator of transcriptioninhaematopoietic
Table1. Expression profilingof coronaryarteries.
Target tissue Experimental treatment Number of genesaffected
Intactcoronaryarteries6 Severe atherosclerosis versus
non-atherosclerotic arteries
56
Diffusesurvey ofseveralarteries7 Documentation of gene expression inatherosclerotictissue
75
Coronary plaquesfrom patients
withstableor unstable angina8 Rupturedplaquematerial 5
Atheroscleroticplaque fromhuman
coronaryarteries9 Specimens retrieved by novelhelixcutter 201
cells.17 Intercellularadhesion molecule 2(ICAM-2)isanother
novelgenewhose expressionisincreased inCAD tissues, and itisexpressedmainlyinendothelialcells.6GSTis theonly
genewhose expressionis reduced inCAD tissues, and it encodesareducing enzyme.18 The most significant group of
genesidentified in thepresentauthors’ study were involved in inflammatory processesand consistedof genesexpressed by B and Tlymphocytes, as wellas macrophages. Othergenes included five genesinvolved in lipidtransfer,oxidationand metabolism; nine genesinvolved incell migration, adhesion andmatrix degradation; 12genesinvolved incell necrosis, apoptosisandproliferation;and severalgenes with currently unknownfunctions.
A cDNAmicroarray study was usedtoanalyse18,376genes forchangesinexpressionin whole-mounthuman athero-scleroticplaquesfrom the internaland externaliliac arteries, aorta,poplitealartery,posterior tibialartery and the tibio-fibular trunk with advancedlesionsascomparedwithnormal arteries.7 Similar to the earlier studybyArchackietal.,6 17
genes previouslyconnectedtoatherosclerosis, egthose coding foractin, cathepsin S,apoEand P-glycoprotein,were identified, whichvalidates microarrayanalysisasavalid approach for studying gene expressioninatherosclerosis.7 The study
identified75new differentiallyexpressed genes, 44of which wereupregulated inadvanced lesions. Differentialexpression ofJanuskinase 1(JAK-1),vascularendothelial growth factor (VEGF)receptor-2andcontinuousdomain-31(CD31)in athero-scleroticplaques wasconfirmedwith reverse-transcription PCR and immunocytochemistry.JAK-1 isaproteinkinase whichmayactivateplatelet-derived growth factor-b (PDGF) signalling.7 VEGFreceptor-2isareceptorforangiogenic
factor VEGF, itactsasanearly marker ofvasculature in embryogenesis.7CD31isalsoanendothelialcell marker thatis
involved in monocyte and T-celladhesion toendothelialcells.7
Thesestudies support the currentauthors’ hypothesis that endothelialdysfunctionisanearly triggering event for the development of CAD.2Thirty-one genes were downregulated
inadvancedlesions,whereas the Archackistudy6 identified
only one downregulated gene indiseased coronaryartery tis-sues.A limitation of the Hiltunenet al. study is that various arteries wereutilised, thus the interpretation of the data may be challenging asdifferent arteries mayhaveunique charac-teristics.7The Hiltunenetal. studydidnot share anygenesin
common withthose found in the current authors’ study, discussed above; thisdiscrepancyingene identification between studiesappears tobe a commonfinding in microarray analysis of atherosclerosis.The potentialexplanationsfor this incongruity maybe thatdifferentarteries (coronaryartery versus otheratherosclerotic tissues) wereused indifferent studies, and that the arteries used were at different stages of disease.
Thethird of the aforementioned microarrayanalyses wasa small-scalestudy with cDNA arrays, in which cDNA probes were hybridisedto nylon arrayscontainingonly 482genes
involved inhaemostasis, inflammationand celladhesion.8
The studyinvestigated gene expressioninhumancoronary plaquesfrom patients withstableor unstable angina.One gene, encoding tissue factor,showed increased expressionin unstable anginasamples,whereasfive genes— including anticoagulant proteinS,cyclooxygenase-1(COX-1),interleukin (IL)-7and the chemokinesmonocyte chemotacticprotein (MCP)-1and -2—were expressed at lower levelsin unstable anginasamples.
In the fourthmicroarray study,rather thaninvestigating coronary arterydiseaseper se, the authorsfocusedon reste-nosis,the mostimportant limitation ofpercutaneous angio-plasty procedures (a common surgical treatmentfor patients with coronaryartery disease).9Zohlnhoferetal. retrieved
tissuespecimens from 16 patients withsymptomatic in-stent restenosis using anovelhelix cutter. The control samples includedmediaspecimens from sevengastrointestinalarteries andsevencoronaryarteriesfrom cardiactransplantation.Atlas cDNA arrays (humancancer 1.2, human 1.2cardiovascular, andstressarrays, Clontech) wereused, and atotal of2,435 genes were examined.The studyidentified severalgenes that hadpreviouslybeen reportedto be expressed in smooth muscle cellsand regulated in neointima and restenosis.These includedupregulation ofthethrombospondin-1(TSP-1),70-kDa heat shockproteinB,COX-1genesand downregulation of desmin.The studyalsoidentified two newgenes thatare connectedto restenosis, FK506bindingprotein-12 (FKBP-12) (upregulated)and the gene encodingthemammary-derived growth inhibitor (MDGI) (downregulated).MDGI isapotent tumour suppressor,19,20and downregulation ofMDGImay
leadto proliferationand migration ofsmoothmuscle cells, resulting in restenosisaftercoronaryinterventions.21
Upre-gulation of FKBP-12 is significantbecause itisinvolved in controllingtransforming growth factor-b (TGF-b) signalling andmay prevent cycle arrest, leadingto there-occlusion of coronaryarteriesby theproliferation ofsmoothmuscle cells.19
This latter study lent support to thetestingof Rapamycin (sirolimus)asanovel pharmacologicalapproachto preventing restenosis, asit binds toFKBP-12and downregulatesTGF-b inhibitoryactivity.22Thepotential useof Rapamycin to
pre-vent restenosisisalso supported by thereport that itinhibits smoothmuscle cell (SMC) migrationandproliferationand intimal thickening afterballoonangioplastyinaporcinemodel ofrestenosis.23
Inaddition to microarrayanalysis,suppression subtractive hybridisation (SSH) technology was successfully usedto study gene expression patternsin whole-mount human stable and rupturedplaques.10,24 The details of the proceduresinvolved
inSSH have beendescribed byFaberetal.10,24Compared
reportedthat ithad a twofold difference inexpressionand was upregulated in rupturedplaques.Asaresult ofthisfinding, it washypothesisedthat the overexpression ofperilipin increases triacylglycerol storage by reducingtriacylglycerol hydrolysis.27,28 Consequently,thisincrease in lipidretention
may leadto plaque destabilisationandrupture.Perilipinwas found tobe consistentlyexpressed ineight ruptured plaques andwascompletelyabsentin stableplaques, identifying itasa uniquemarker.Inaddition, fibronectinand immunoglobulin lchain were downregulated in rupturedplaques.
In summary,thestudiesdiscussed above highlight the importanceofusing microarrays toidentify genes with potential rolesin the generation of atherosclerosis, including the evolution ofplaque andplaquerupture.Novelgeneshave beenidentified whichmay serve asfocal points of future functionalandpharmacological studiesinaneffort toalter the process of atherosclerosis. Furthermore,the combination of microarrayanalysisand positionalcloningmay provide a powerful synergyin the characterisation ofthemolecularbases of humandiseases. First,the genesidentified by microarray analysisbecomethestrong candidate genesiftheyarelocated inapreviously mapped geneticsusceptibility locus for the disease.Secondly,theresultsfrom microarrayanalyses may provide independent supportive evidence for thenew signal-lingpathwayfor the pathogenesis ofthe disease identified by positionalcloning.Anexcellentexample is the authors’ recent identification ofthe first non-lipid-related disease-causing gene,myocyte enhancer specific factor-2A(MEF2A), forCAD and MI.2 Using alarge family with13 patients whodisplayed an
autosomaldominant inheritancepattern of CAD and MI, Wang etal.successfullyidentified a significant linkageto CAD/MIonchromosome15q26 (LODscore 4.19),the first locus forautosomaldominantCAD and MI (adCAD1).The adCAD1diseaselocus contains 93 known or putative genes, and mutationalanalysisestablishedoneofthe genesin the region,MEF2A, as the generesponsible forCAD and MI.A 21basepairdeletion ofMEF2Aresultsin the deletion ofseven aminoacidsin the MEF2A protein (D7aa), and co-segregates withthe disease in the family. It reduces thetranscriptional activationactivity of MEF2A byapproximately threefold and abolishes thesynergistic activation oftranscriptionbyMEF2A incombination withthe Gallus gallus GATAtranscription factor-1 (GATA-1) through a dominant-negativemechanism. Using immunostaining, a strong MEF2Aprotein signal was detected incultured endothelialcellsand also within the endothelium of coronaryarteries,which isa barrierbetween vessels and blood elements such as the plateletsand macro-phages that are central to the pathophysiology of CAD.The authors’ studies suggest that anearly step in the development of CAD/MImay involve deregulation ofspecific transcrip-tional programmesin the endothelium.Defectiveor abnor-mallydevelopedvascularendothelium willbemoresusceptible toinflammationand the diapedesis ofmonocytes, and expose the subendothelial matrix to the genesis of atherosclerotic
plaqueor thrombosis.Consistent withthe authors’hypothesis, microarrayanalysis of CAD, atherosclerosisandrestenosis revealed differentialexpression patterns ofseveralgenes involved in vasculogenesis, angiogenesisandvascular remo-delling, includingextracellulargrowth factor-1(ECGF1)and MMP-9in thestudybyArchackietal.,6VEGFreceptor 2, JAK-1
andCD31in thestudybyHiltunenetal.,7TSP-1in thestudy
byZohlnhoferetal.,9andMCP-1andMCP-2in thestudyby
Randietal.8Itisanticipatedthat microarrayanalysis willbe
increasingly used in thepositionalcloningstudiesfor identifi-cation ofsusceptibilitygenesforcomplexhumandiseases.
E
xpr
e
ss
i
on pro
fi
l
i
n
g
o
f i
s
chae
m
ic
myo
ca
r
di
um
i
n
a
n
i
m
a
l mo
de
ls
Alimitation ofusing human tissuesfor microarrayanalysisis that there isatremendousamount of biological variability. Astudy of human tissuesfrom thepopulation usuallycontains patients withseveral medical problems,taking several medi-cationsand withprior risk factorsforatherosclerosis.A study using animal modelscan overcomethe problem of biological variability.Althoughvery limitedtodate, gene expression profiling hasbeen performedon myocardial tissuesin several animal models thathave been usedto provide information on theprocess of MI ina controlledsetting, enablingthestudy of the gene expression profileof MI.Theprecisemechanisms of ischaemic injury to themyocardium secondary to athero-sclerosishavenot beenfullyelucidated.Withmicroarray technology, however,one canbegin tohave a comprehensive overview ofthe gene expression patterns occurring in the ischaemic heart (Table2).
Rabbithearts that weresubjectedto two five-minute episodes of ischaemia(followed byfiveminutes ofreperfusion) and then tofive hours of additional reperfusionidentified 35 genes that were differentiallyexpressed.29A strong
upre-gulation ofmitogen-activatedproteinkinase-activatedprotein
kinase-3(MAPKAPK-3) wasidentified.It wasinferredthat thisgene hasaprotectivemechanismagainstcellinjury, asa related gene,MAPKAPK-2, isknown tobe atheroprotective.30
Similarly,thesame group studied the samemodel of ischaemia in rathearts subjectedtoa 20-minutetransient episodeof ischaemia followed by fourhours of reperfusion.31
Thepurposeofthestudy was todeterminewhetheractivation ofprotective genes takes placewithinfourhoursfollowing a brief episodeof ischaemia,mimicking anginapectoris.31
Stantonetal. studied anin vivoratMImodelbycausing a permanentcoronary occlusion.32Onamicroarray with 4,000
probes,theyfound atotal of731genesdifferentiallyexpressed duringthe16-week experimental period from two regions of the heart,theleft ventricularfreewallandthe interventricular septum.Upregulated genesincludedthose encoding atrial natriureticpeptide(ANP),sarcoplasmic/endoplasmicreticulum Ca2þ-ATPase, collagen, fibronectin, decorin, fibulin,tissue
inhibitor ofmetalloproteinase-3, fibrillin,laminin,secreted proteinacidic andrich incysteine(SPARC)and osteoblast-specific factor-2.These genes were classified asbelongingtoa remodellingpathwayin thepost-MIperiod.The functional clusteringofthe genelist showedthat themajority ofthe upregulated genes were classified ascytoskeletaland extracellular matrix (ECM) proteins,while downregulated genes were classified ascontractileproteins orfattyacidmetabolism-related genes,suggesting aprofound change in the energy-generating processesin the ischaemic andtheninjuredmyocardial tissue.
In thesettingof MI,the angiotensin-converting enzyme inhibitor, captopril,wasadministeredto ratsforeight weeks after ligation oftheir leftcoronaryartery.33Incontrolanimals,
therewasanincreased expression of collagenI and III, throm-bospondin-4,lysyl oxidase, fibronectinand biglycaneight weeks afterMI.Therewasalso upregulation of components ofthe complement pathwaysandlipopolysaccharide bindingproteins and VCAM-1.The downregulation of fattyacidmetabolising enzymes wasalsodocumented.With captopril treatment, therewasa downregulation of hypertrophy-related genes such asTGF-b3and insulin-like growth factorbindingprotein-6.These resultsindicatethat pharmacological treatmentin thepost-MI period canhave animpact ongene expression. Manygenes werenotaffected, however,suggestingthe need for more novelforms oftherapy toalterischaemic damage afteranMI.
Finally, another rat modelemployed to studygene expressionin themyocardium, induced withtheligation of
the left anteriordescending (LAD)coronaryartery, docu-mented gene expressionafter 24 hours.34 Severalgenes
dif-ferentiallyexpressed included genesassociated with cardiac muscle development such as the cellcycleregulator p18ink4 andthestructural proteins,a-myosin heavychain (a-MHC) andfetal myosinalkalilightchain (MLC).Expression of early growthresponse factorsEgr-1and Egr-3wereupregulated by ischaemia,whereas therewasa downregulation ofGST.Other stress responses toischaemia included aninductionin the expression of the apoptosis regulator Bax, whichmay con-tributeto celldeath.Thisgenelist may representa balance between the cardioprotective and degenerativeprocesses that accompany myocardialischaemia.
In summary,microarrayshave been utilised to understand the consequences of atherosclerosis,which canabruptlycut off the supply of blood andoxygen to the myocardium.The microarrayenablesinvestigators to look at a comprehensive overview of genetic changesand may stimulatethe develop-ment ofpharmacological therapy toalter the processes involved in the consequences of MI. It isinteresting to note that severalgenes were identified byboth the humanand animal model studies (genesfor 70kDa heat shockprotein, fibronectin, VCAM-1 and GST),suggestingthat microarray analysis of animal modelsisappropriate for some aspects ofthe characterisation of humandiseases.
E
xpr
e
ss
i
on pro
fi
l
i
n
g
o
f
e
n
d
ot
he
l
ia
l
ce
lls
The endothelialcell layer of anartery is the first strategic locationfor the initiation of atherosclerosis. Dysfunction and/or injury of endothelialcells playanimportant role in atherogenesis.2The endotheliumhas manyfunctions —
forexample,regulation oftone, coagulationand
Table2. Expression profilingof ischaemicmyocardiuminanimal models.
Target tissue Experimental treatment Number of genesaffected
Rabbitheart29 Twofive-minute episodes of ischaemia followed byfive hours ofreperfusion
Strongupregulation of MAPKAPK-3
Rathearts30,31 20-minute episodeof ischaemia followed byfourhours ofreperfusion
Significant upregulation of HSP-27and70
Ratin vivomyocardial
infarction model32 Permanentcoronary occlusion 731differentiallyexpressed genes Rats’hearts33 Administration of angiotensin-converting
enzyme(ACE)inhibitor
37genesclustered into 11functional
groups, ACE inhibitionafter myocardialinfarction
inhibitscardiac hypertrophy
Rats’hearts34 Ligation oftheleftanterior
descending coronaryartery
fibrinolysis— and it secretes several substances. Expression profiling hasbeen performed forendothelialcell lines to study the impact ofoxidised low-density lipoprotein (LDL), regu-lators of inflammation (tumour necrosisfactor-a [TNF-a], IL-1b), infection withChlamydiapneumoniae(CP), antioxi-dants, homocysteine and differential patterns of blood flow. Itishypothesisedthatareas ofthevascular system with turbulentflowaremorelikely todevelopatherosclerotic plaques thanareregions withlaminarblood flow.35Expression
profilingwas usedtoexaminethe differentialexpression of genesinhumanaortic endothelialcells (HAECs) that were exposedto thesetwoconditions (Table3).36In total,more
than 130genes were induced by turbulentflowascompared withlaminarflow.Approximately50 percent ofthe genes surveyed did have a detectable expressionat baseline, butat twohours 3–8percent ofthe expressed genes were upregu-lated, and by 24 hours 14.2 percent—or50 genes—were downregulated.Thestudydemonstratedthat changesinflow serve asa distinctbiomechanical stimulus which hasaprofound impact upon the gene expression profileof HAECin vitro.
LDL is wellestablished ascontributingto the generation of atherosclerosis.Oxidation of LDL isa keyevent associated with endothelialcellinjury, by leadingto the expression of endothelialcell receptors that bindtoinflammatory mole-cules.37Primary human umbilical veinendothelialcells
(HUVECs) were exposedto oxidised LDL for sixhours.38
Ofthe 588 genesevaluated,78 genesdisplayed a greater than twofold change inexpression levels: 57genes wereupregulated and 21genes were downregulated in responseto theoxidised LDL. Oxidised LDLsignificantlyaffectedthe expression of genesencodingtranscriptionfactors, cell receptors, adhesion molecules, ECMproteinsand enzymesinvolved incholesterol metabolism.It isinterestingto notethatexpression ofretinoic acidreceptor-b(RXR-b) was upregulated inboththeturbulent flow and oxidised LDL studies.
Gene expressionhasbeendocumented inendothelialcells infectedwithpathogens such ashumancytomegalovirus (HCMV) orCP.Thesepathogens arelinkedto the
develop-ment of vasculardisease, including atherosclerosis,39 yet the
roleof pathogens in vasculopathieshasbeencontroversial. Expression profiling hasidentifiedmechanismsby which CP alterscoronaryarteryendothelialcells.40Twenty of268 genes
on the array wereupregulated inhuman endothelialcellsin responsetoCP,suggestingthatCP infectiondoes not leadtoa generalisedresponse ingene expression. Genescoding for cytokines (IL-1), chemokines (MCP-1and IL-8)and cellular growth factors (heparin-binding epidermal-like growth factor,basic fibroblastgrowth factor(FGF)andPDGF-bchain) werethemost prominently upregulated in responseto the atypicalbacteria. Additionally, increasesin the expression of genescoding for intracellularkinasesand cell surface receptors withsignal transductionactivities wereobserved.Time course exper-iments showed that mRNA levels wereupregulated within twohoursfollowing infection.
Novelgrowth factorgenes were also identified and included b-FGF,HBGF,PDGF-band activinA.b-FGF suggested apathwayincommon with endothelialcellsand SMCs.HBGF isa growth factor synthesised byendothelial cells which canactasapotent mitogenfor theproliferation of SMCs.41Similarly, PDGF-bnot only hasSMC
growth-promoting activity but isalsoassociatedwithneointimal proliferation of SMCs42 PDGF-b isalso a growth factor
accessory moleculethat modulates the activity ofspecific growth factors.42ActivinA,orerythroid differentiation
pro-tein, hasbeen shown to modulatemonocyte/macrophage function, including immunologicalactivation ofmonocytes43
andthe induction of MMP-2.44CP inducedthe expression of
E-selectin,ICAM-2andVCAM-1twohoursafter infection, yet returned tobasal levelsby 24 hour post-infection;45 these
genes were also upregulated in responseto oxidised LDL. E-selectinand VCAM-1were also upregulated in the endo-thelialcellsexposedto laminar ordisturbed flow.Theseresults identify theresponseof endothelialcells toCP bydefiningthe list of CP-inducible genesand providenewinsightsinto potential mechanisms of atherogenesisfrom these intracellular bacteria.
Table3. Expression profilingof endothelialcells (ECs).
Target tissue Experimental treatment Number of genesaffected
Aortic EC36 Disturbed flowandsteady laminarflow 100 Coronaryartery38 Oxidisedlow-density lipoprotein 78
Coronaryartery40 Infection withChlamydiapneumoniae 268
Coronaryartery46 Homocysteine 600
Coronaryartery48 Nicotine 4
Umbilical vein49 BO-653,probucoland bento(h)quinoline(BHQ) 17 Smoothmuscle cells (SMCs)from plaque50 SMCsþtumour necrosisfactor (TNF)-a 5
Several otherfactorsdirectlyaltergene expressionin endothelialcells.Elevated homocysteinelevels have been identified asarisk factorforCAD and have been shown toalter gene expressioninendothelialcells.46Usingthe cardiovascular
cDNAmicroarrayapproach,the expression of600genesin endothelialcells was screened and asubset wasidentified and consideredto bemodulated by homocysteine.47 They were
clustered accordingto the function of the encoded proteins, such as: endothelial motility,signalling and lipidmetabolism. Similarly,oligonucleotide arrays wereused toidentifygene expressioninendothelialcellsexposed to nicotine,themajor constituent of cigarettesmoke.48Themost significantchanges
ingene expressionfound weretheupregulation of proteins in the inositol phospholipid pathway,namely phosphatidyl-inositol phosphate kinase and diacylglycerol kinase.Changes were alsodetected for transcriptionfactors, cAMPresponse elementbindingproteinand nuclearfactor (NF)-kappa-B.
Takabeetal.examined gene expressioninendothelial cellsexposed toBO-653,probucoland bento(h)quinoline (BHQ)—which actasfree-radical scavenging antioxidants.49
These agents were initially developed asanti-atherosclerotic medications.Among6,416genes,17genes— includingthose encodingmitochondrial proteins and proteins relatedto oxidativestress responses—were inducedmorethan threefold by thesethree drugs.By contrast, genes of threesubunits of the proteosome(PSMA2,PSMA3,PSMA4) were downre-gulated.The gene coding for the cytochrome P-450 1A1 isozymepathway, a drugmetabolisingphase I enzyme, was expressedonlybyBHQtreatment.
Cultures of coronaryarteryendotheliumand SMCsderived fromasingle coronaryarteryhave beenexposedtoIL-1band TNF-a,potent stimulators of inflammation.50 The most
noticeable difference between the cell types wasa considerable greater magnitude and complexity of the transcriptional response in the endothelialcells.Amongthe209 regulated genesin the endothelium,99 werenot previously linked directly to atherosclerosisincludingthosethathad been associatedwithleukocyte function (eg, IL-7 receptor, EBI-3 receptor)andothers relatedtoantiviraland antibacterialdefense (eg, oligoadenylatesynthetase,LMP7, toll-likereceptor 4, comp
-lementcomponent 3).Inaddition, 43 genes likely to participate in signal transduction (egIL-18receptor,STK2kinase, STAF-50,ANPreceptor,VIPreceptor,RAC-3,IFP-35) were expressed. This providesevidencethat amajoreffect of TNF-aand IL-1bis toalter thepotential ofthe endothelialcell to respond to various otherexternal stimuli.
In summary,multiplepathwaysare altered inendothelial dysfunction.Expression profiling hasdocumentedthe altera-tionsinexpression of groups of genesby manycommonagents includinglaminarflow, infectiousagents such asCP andtoxic agents like homocysteine,oxidised LDL andnicotine. Altera-tion ofthe function of endothelialcells may serve as the first strategiclocationforhaltingthe cascadeof events that may follow modification of gene expressionin these cells.Each
experimental protocoldiscussed above elicited analterationin the expression of aunique cluster of genes.Althoughmost genes werenotfoundtobe commonamongthestudies,this maybe attributedto theuniqueresponseofthe endothelialcell under thevariousconditions.Nevertheless,this may provide further medical strategiesandtargetsfordownregulatingthe inflammatory responsewhichultimately leads toatherosclerosis.
E
xpr
e
ss
i
on pro
fi
l
i
n
g
o
f
v
a
s
c
ul
a
r
SMC
s
The proliferation, migrationand invasion of SMCs through-outa coronaryartery make asignificant contribution to atherosclerosis. Lateocclusion of autologous saphenous vein grafts (SVGs)isdueto medialandneointimal thickening secondary to migrationand proliferation of SMCsandthe subsequentformation of atheroscleroticplaques. This process is themaincauseofrestenosisafter percutaneous transluminal coronary angioplasty (PTCA).Understanding SMC prolifer-ation may provide insightsinto thepathogenicmechanisms of CAD, as wellas restenosisafter PTCA.Expression profiling of SMCshasidentified alterationsingene expression that may berelatedto thetransition ofquiescent, contractile SMCs to proliferative SMCs.
Human vascularSMCs were exposed toiloprost, a prosta-cyclin (PGI2)analogues which activatescyclic adenosine
30,50monophosphatesignalling,which isassociatedwith
maintaining SMCsinaquiescent state(Table 4).51 PGI 2 is
known tobe atheroprotective, asithasbeen shown toexert manyeffects such as vasodilation, inhibition ofplatelet aggregationand modulation of cholesterol metabolism,52 as
well as preventing SMC proliferationand migration.53,54
Atotal of 83genes were differentiallyexpressedsixhoursafter iloprostexposure.Several ofthese genesaccountfor the atheroprotectiveresponseto iloprost.Mitogenactivated protein (MAP)kinasephosphatase-1 (MKP-1)hasbeen regarded asa criticalcounteracting factor for several tran-scriptionfactors (p38, c-Jun-N-terminalkinase [JNK], extracellular-signal related/regulated kinase [ERK]), leading to the downregulation ofstimuli inducingtheproliferation of SMCs.55 Thezinc finger protein, hEZF, isassociatedwith
growth arrest, asit hasbeen shown toinhibit DNAsynthesis infibroblasts overexpressingthisgene.56The gene,Cyr61,was
found tobe downregulated byiloprost.Thisgeneplaysarole in mediating celladhesionand migrationand augments growth factor-induced DNA synthesisinfibroblasts.57 Other
criticalgenesinclude: has2(of uncertain physiological conse-quence),stanniocalcin (cell protectionagainstischaemia58),
plasminogenactivatorinhibitor-1(itsdownregulation mayenhance fibrinolytic activity59),MCP-1(itsdownregulation may
pre-vent monocyte attachment toSMCs60), hemeoxygenase-1
(inhibitsSMC proliferation61)and COX-1(promotes the
mechanismforenhancing prostaglandin levels62).
Downregu-lation ofMCP-1andCOX-1wasidentified inatherosclerotic plaquesfrom human patients withunstable angina.8
Microarrayanalysis using 558 cardiovascular-associated genesidentified 47 genesdifferentiallyexpressed in prolifer-ating andnon-proliferating humanarterialSMCs.63Most of
the genesin thestudy were associatedwiththe ECM and cell motility.Themaingene groupsidentified included matrix-organisingproteins, ECMproteins, celladhesion proteins, and extracellularcommunicationand cytoskeleton motility pro-teins. Genes previouslyassociated with SMCmigrationand invasion,such as those coding for tissue inhibitor of metallo-proteinases (TIMP)-2, TIMP-3and MMP-3,were confirmed by the arraydata.Reduced expression ofseveralcytoskeletal proteins,such as vimentin, fibronectin, cytokeratins and b1-integrin,were downregulated in the invasivephenotype. Furthermore, angio-associatedprotein,a-E-cateninand brain ANPreceptor were downregulated,whereas tissue factor pathwayinhibitor-2 (TFPI-2) was strongly upregulated in invasiveproliferating SMCs.Consequently,the data document the expression profileof essentialgenesinSMCsinvolved in the invasiveprocess.
Microarrayanalysis with 8,600genesidentified.20-fold increasesin steady-state eotaxin mRNA.Moreover, follow-up immunohistochemical studies withtissuesamplesfrom seven non-diseased and 14 atherosclerotic arteriesdemonstrated overexpression of eotaxin proteinand its receptor, CCR3, in cultured humanaortic SMCs treatedwith TNF-a(which induces vascular inflammation), whereas theirexpression was negligible in normal vessels.64This was the first study to report
the expression of eotaxinbyhumanatheroma.Whilethis was known to be apotent eosinophilchemoattractantand activa-tor,65,66noeosinophils were identified in the target tissue.
Similarly, CCR3had previouslybeen observed in macro-phages,mastcells,neutrophilsand endothelialcellsin endo-bronchialbiopsies of the atopic asthmatic lung.67The
implications ofthe studybyHaleyetal.arethat TNF-amay recruit and activatemacrophagesandmastcells throughthe CCR3 receptor.64 This study may leadto the generation of
eithereotaxin-orCCR3-null mice,whichmightberesistant toatherogenesis.
Inaddition toSVGsbeingused forcoronaryarterybypass grafting,leftandrightinternal mammaryarterieshave also been used.68 The SVG is thestandard conduitforcoronary
arterybypassgraftingtoallareas ofthe heartexcept the LAD coronaryartery.Accelerated atherosclerosis occurs in venous conduits suchthatduringthe first yearafterbypass surgery, 15percent ofveingrafts occlude;between one andsix years, the graftattrition rate is 1–2 percent per year;and between six andten years, itis4percent per year.69Thus,within the first
decadeof bypass surgery,mostbypassgrafts willbeoccludedor significantlydiseased.Amajorcomponent ofthis occlusionis theproliferation of SMCs.Differentiallyexpressed genesin thesaphenous veinhave beendocumented and correlatewith expected gene expression patterns, includingupregulation of c-Junand CDK-10.Inaddition,previously unidentified gene expression patterns were detected,such as theupregulation of HSP-70,fibronectin-1,erbB-3 proto-oncogeneandc-myc.
The effect ofa-tocopherol treatment ongene expressionin humanaortic SMCshasbeen studied because it mayaccelerate woundrepairandtissueregenerationduring atherosclerosis.70
This medicationisalso often recommended becauseof its anti-oxidant properties.71The expression oftheconnectivetissue
growth factor(CTGF)genewasinduced by a-tocopherol 1.8-fold.It washypothesised that thisgene isinvolved in normal repair processesand is permanently overexpressed in patho-logicalevents.70a-tocopherol mayeither stimulatethe
syn-thesis of CTGF or interferewiththe effect of TNF-aon downregulation ofthisgene.
cDNA array technology hasbeen utilised to unravel the molecularbasis ofthe antiproliferative effect of butyrateon vascularSMCs. Butyrate isanaturalfattyacid ester which has been shown toinhibitSMCproliferationinin vitrocellculture systems.72It isderived from themicrobial metabolism of
dietary fibre in the colon,where it is produced inhigh levels.73It canfunctionasananti-inflammatoryagentby
suppressing intestinalinflammation,74 and mayaltergene
expressionby reversiblyinhibiting histone deacetylase, causing the hyperacetylation of histonesand thereby leadingto selec-tive changesingene expression.73Toassess the involvement of
gene expressioninbutyrate-inhibitedvascularSMC prolife-ration,proliferatingvascularSMCs were exposedtobutyrate.75
Table 4. Expression profilingofsmoothmuscle cells (SMCs).
Target tissue Experimentaldesign Number of genesaffected
Human vascularSMCs50 Exposureof cells toiloprost 83 SMCsfromcoronaryartery63 Quiescentand invasive SMCs 47
Humanaortic SMCs64 Exposureto tumour necrosisfactor-a Focus oneotaxinand CCR3 receptor
Saphenous veingrafts68 Comparison withungraftedveins 6
Humanaorta70 a-Tocopherol treatment Focus onconnectivetissue growth factor
Atotal of111genesexhibitingmoderate(two-tofivefold) to strong(.fivefold)differentialexpression were identified. Analysis ofthese genesindicates thatbutyratetreatment mainly alters the expression of fourdifferentfunctionalclasses of genes, including 43genesimplicated incell growth and differentiation,13genes relatedto stress response,11genes associatedwithvascularfunctionand eight genes normally presentin neuronalcells.cDNA expression profilesindicate that butyrate-inhibitedvascularSMC proliferationinvolvesa combined action of aproportionally largenumber of both positive and negativeregulators of growth,whichultimately causesgrowth arrest of vascularSMCs.75Forexample,the
downregulated genesincludedpRb(required forG1 toSphase entry), Sphase-specificmus musculus podocan(PCAN),G2/M
phase-specific cyclinB1andcdc2, andp55cdc.The downregulation ofthese critical positiveregulators of cell cycleprogression indicates that butyrate exerts its antiproliferative effectby altering key positiveregulators of all phases of cellcycle pro-gression.Furthermore, butyrate alsoappeared toblock SMC proliferationby upregulatingnegativeregulators of cellgrowth ordifferentiationinducers such asp21waf1, p14INK4B/ p15INK5B and clusterin. Consequently,the identification of the molecular mechanisms of butyrate-inhibitedproliferation isimportant,not only to understand the molecularbasis of butyrate-induced SMCproliferation, but alsobecauseof its potential use asatherapeutic agent to prevent restenosis of arteriesinhumans.
In summary,smoothmuscle cell proliferationcontributes to the formation of atheroscleroticlesionsandrestenosisafter angioplasty, andmicroarray studieshave identifiedmultiple genes thatare involved in this process.Understandingthe molecular mechanismsinvolvedmay providenovelinsightsinto pharmacologicalapproaches to preventing SMCmigration.
E
xpr
e
ss
i
on pro
fi
l
i
n
g
o
f
i
n
fla
mm
a
tory
ce
lls
Inflammatorycells playanimportant role in the development of CAD.First,monocyterecruitment isa keyeventin the
formation ofthe earliest vascular lesion, the fatty streak. Secondly,macrophageuptakeofmodified LDLs viascavenger receptors gives risetofoamcellsin the atheroma, andthese secrete growth factors, cytokinesand inflammatory mediators thatinfluencethe growth and development ofothercell types within the atherosclerotic lesion, generatingplaques. Micro-arrayshave been usedto profile gene expressionchangesin the inflammatory cellsin relation to the formation of athero-scleroticlesions.
Gene expression ofmacrophageshasbeenanalysedusing microarrayscontaining approximately 16,000human
cDNAs (Table 5).76The human monocyticleukaemia cell line
(THP-1) was treatedwithoxidised LDL.The cells werethen activated by treatment withlipopolysaccharide(LPS), and RNAwasharvested at zero,one and six hours afterLPS addition. Oxidised LDL treatment affectedthe expression of 57 of127genesin macrophages. Among them are genes that code for potentintracellularand extracellular signalling molecules such asNF-kB, A20andnumerouscytokinesand chemokines. Oxidised LDL pretreatment resulted ina signi-ficantchange inLPS-induced NF-kB activation.Someofthe oxidised LDL effects weremediated by the nuclear receptors RXR and peroxisomal proliferator-activatedreceptor-g.
Tofurtherdefinethe mechanismsby which LDLparticles affect macrophagesand foamcells, alarge-scale gene expressionanalysis of cholesterol-loaded macrophages was carried out.77 Oxidised LDL-treated and time-matched
con-trol untreated cells were hybridised to microarrayscontaining 9,808 humangenes.Twohundred andsixty-eightgenes were found to be differentiallyexpressed at least twofold at oneor moretimepoints.Thesepatterns ofregulation were classified into sevenclusters.Shiffmanet al.identifiedpatterns of gene expression of knownandnovel molecularcomponents ofthe cellular responsethat are implicated in the growth,survival, migratory, inflammatoryand matrix remodelling activity of vessel wall macrophages.77 The data indicatethat oxidised
LDLloadingof THP-1 cellsdidnotelicit any measurable immediately transcriptional response from the genes studied untilday4. Thenoveland most highly upregulated genes identifiedwere: DSCR1, liverand activation-regulated chemokine
Table 5. Expression profileof inflammatorycells.
Target tissue Experimentaldesign Number of genesaffected
Macrophages76 Oxidisedlowdensity lipoprotein 268 Human monocyticleukaemia cell line77 Lipopolysaccharide 57
Macrophages78 Exposuretocopper 7
Macrophages80 Exposuretoarsenic 62
THP-1cells81 Biomechanicalforces 3
Macrophages86 Exposuretoandrogens 27genesexclusively
(LARC)/MIP-3a,CD73/50-nucleotidase,epithelial membrane protein-1,uridinephosphorylaseandtwoexpressedsequencetags (ESTs). The incidenceofthese genesindicates that the pre-viously published dataon oxidised LDLloading of macro-phages only partiallydescribes the changes that occurin some inflammatorycells.The mosthighlydownregulated genes included promoters ofthe anti-microbial potential of macro-phages (carbonic anhydrase,cytochrome b-245,RNase A2,CD64) and three involved incellcycleprogression or synthesis of precursorsinvolved incell division (genomicoryza saliva protein[GOS2]protein, thymidylatesynthetase,laminB1).
Similarly, DNAmicroarrays wereused todefinethe changesingene expression profile in responseto copper exposureof human macrophages.78Serumcopperhasbeen
shown tobe associated with cardiovasculardisease(a3.5-fold increasedriskof acute MI)butits mechanism of actionhas not beenidentified.79 Theresults showedthat expression of 91
genes were altered.Copper increased the expression ofseven cholesterogenic genes (3-hydroxy-3-methylglutarylcoenzyme A
synthase,isopentenyl pyrophosphate isomerase,squalenesynthase, squalene epoxidase,methyl sterol oxidase, H105e3 mRNA and sterol-C5-desaturase)and the gene encodingtheLDLreceptor, and decreased the expression ofCD36 andlipid-binding proteins. Thus, copperactivatescholesterogenic genesin macrophagesinvolved inLDLuptake and denovocholesterol biosynthesis. This leads to lipid accumulationin the inflam-matorycellsandthus the arterial wall.
Gene expression wasalso studied in lymphocytesexposed toarsenic,which isassociated with anincreasedrisk for vas-culardisease.80 Patients were exposedto low, intermediateor
highlevels of arsenic asdetermined by monitoringserum levels.Atotal of62genes showed asignificantdifference in the intermediate and highlevels of blood arsenic, ascompared withlow levels. These genesincluded those encodingIL-1b, IL-6,chemokine C-Cmotifligand2/MCP-1,chemokine C-X-C
motifligand1/growth-relatedoncogene-a,chemokine C-X-Cmotif
ligand2/growth-relatedoncogene-b,CD14 antigenand MMP-1. The effect of biomechanicalforces on macrophage func-tionsinatherosclerosis wasdetermined by microarrays with 1,056genes.81Mechanicaldeformationat 1Hz wasappliedto
athin transparent membraneon whichthe cells were cultured. Thisinduced only three genes to morethan 2.5-fold higher expressionat three andsixhours:prostate apoptosis response-4 (3.0-fold at three hours,6.7-fold at six hours),IL-8(4.3-fold at sixhours)andtheimmediate-early responsegene,IEX-1 (2.6-fold at sixhours).IL-8maybe importantin the initiationand amplification of inflammationand angiogenesisin athero-sclerosis, because itischemotactic for vascularSMCs, T lymphocytesand neutrophils.82 It also triggers the firm
adhesion of monocytes to thevascularendothelium.83 IEX-1
isanNF-kB-inducible immediate-earlygenewhich inhibits apoptosis.84 It mayalso participate in the differentiation of
monocytes/macrophages. Protease-activatedreceptor-4 (PAR-4)isknown tobe a widelyexpressedprotein which
confers sensitisation to apoptosis-induced insults.85
Conse-quently, Johnstoneetal.demonstratedthathuman monocytic cells respondto mechanicaldeformation with induction of immediate-earlyand inflammatorygenes.This suggests that mechanical stressin vivo, such as that associatedwith hyper-tension,may playanimportant partinatherogenesisand instability of coronaryartery plaques through biomechanical effects on vascular macrophages.
Human monocyte-derivedmacrophagesfromhealthy male and female donors have beenexposedto androgen (dihydro-testosterone)inaneffort todetect gender-specific changesin gene expression,86 asithasalreadybeenestablishedthat there
isa genderdifference in theseverity of disease, withmen havingmoresevere coronaryarterydiseasethan women.87 In
macrophagesharvested from males, treatment with androgen resulted in the differential upregulation of27genes,whereas nonewas upregulated infemale harvestedmacrophages.Some ofthese genesdirectly relatedto theprocess of atherosclerosis, including the upregulation ofacylcoenzyme A:cholesterolacyl transferase Iandlysosomalacidlipase(LAL)—whichplayarole in the delivery oflipoproteins tocells,88caveolin-2, CD40,
leukotriene B4 receptor and cadherin-19(the latter threeplaya role in inducing cell surfacereceptors for monocyte attach-ment89). Functional studies tocomplement the microarray
results showed a direct clinicalcorrelation with LAL, in that therewasa consistent increase inactivity with125I-AcLDL
exposure.88 It canbe concludedthat androgenexposure asit
occurs naturallyin themale gender isdirectly responsible for inducing genesassociatedwiththe development of athero-sclerosisand mayaccountforgenderdifferencesin the inci-denceof CAD.
E
xpr
e
ss
i
on pro
fi
l
i
n
g
o
f CHF
CHF isa term used todescribe anyconditionin whichthe heartis unabletogenerate anadequatepressureto pump blood throughout the body. Thisconditioncauses symptoms such as shortness of breath(dyspnoea), fatigue,weakness and swelling(oedema) ofthe legsand sometimesin the abdomen (ascites).Anestimated 4.8millionAmericanshave CHF,90and
halfof all patientsdiagnosedwith CHFwill be deadwithin fiveyears.Eachyear,there are anestimated700,000 newcases in the USA.Increasingprevalence, hospitalisationsand deaths havemade CHF amajorchronic conditionin the USA.It often occurs as the endstageof cardiac disease,referredtoas ischaemic cardiomyopathy (ICM)dueto severe CAD. Expression profilingof humancardiactissueshasbeen employedtoidentifygenes whose expressionis linkedtoCHF, and thesestudiesaresummarised below.
Oligonucleotide microarrays wereusedto profileseven non-failing (NF)and eight failing(F)humanhearts with a diagnosis of end-stage dilated cardiomyopathy (DCM) (Table6).91 Of6,606 genes on the microarray,103genesin
and NF hearts.InF hearts, Kmeans clustering revealedtwo potentially novel pathwaysassociatedwithup-regulation of atrial natriuretic factorandbrain natriureticpeptide andwith increased expression of ECMproteins. A dendrogram or genetic profileofthetwo tissuesdistinguishedtwoF hearts with distinctaetiologies (familialand alcoholic cardiomyopa-thy,respectively).The studydemonstratedthat there isa uniquemolecular signature inF hearts.
Similarly, a‘genomicportrait’ of heartfailure derivedwith microarrays was obtained from end-stage DCMusing the CardioChip withnon-redundant 10,848-element human cardiovascular-based ESTs.92Morethan 100 transcripts were
consistentlydifferentiallyexpressed inDCM.1.5-fold(versus pooled NF hearts).ANPwasalsofoundto beupregulated in DCM(19-fold comparedwith NF hearts)as wellas numerous sarcomeric and cytoskeletal proteins (eg cardiactroponin, tropomyosin),stress responseproteins (eg HSP-40, HSP-70) andtranscription/translation regulators (eg CCAAT box binding factor, eIF-1AY).Downregulated genes were classified ascell-signalling channels and mediators, particularly those involved incalcium pathways (calcium/calmodulin-dependent
kinase,inositol 1,4,5-trisphosphate receptor and sarcoplasmic
reticulumCa2þ-ATPase[SERCA]).Several novel,
cardiac-enriched ESTs were alsoco-expressed.Thestudydemonstrated a commonexpression patternamongthesets ofsamples with DCM and documentsa genelist that may serve as possible targetsfor therapeutic intervention specifictocardiactissue.
Also usingpatients with eitherDCMorhypertrophic cardiomyopathy (HCM)atend-stage CHF, Hwangetal. generated alist ofpredictivemarkersfor thesetwo diseases.93Similar to previous studies, ANP and SERCA
were foundtobe increased and decreased,respectively, and this wasconsistentinall ofthetissues studied.Othergenes havingthesame expression levelsinboth DCM and HCM tissuesincluded:copper/zincsuperoxide dismutase,heat shock
protein 90,elongationfactor-2,calcium-activatedneutral protease, decorinandCD59.Genesfoundtobe differentiallyexpressed betweenDCM and HCM includedatrial myosinalkalilight
chain,calsequestrin,lipocortinandlumican,whichwere upregu-lated,whilemyctranscriptionalactivityandb-dystrobrevin were downregulated.In total,thestudydocumented192genes. Asin theprevious studies reviewed,ANPandSERCA displayedthesame expression patternsconsistentlyinboth populations ofsamples.
Table6. Expression profilingof congestive heartfailure(CHF).
Target tissue Experimentaldesign Number of genesaffected
End-stage dilated
cardiomyopathy (CM)91 Failingversus non-failing 103 End-stage dilated CM92 Failingversus non-failing 100
CM93 Dilatedversusischaemic
myocardial tissue
192
Humanheart94 Fourgroups: i) left ventricle(LV);ii)LV from
failing heartsaffected by
dilated CM);iii)LV fromfailing heartsaffected byischaemic CM;iv) right ventricle
1,306
Humanheart98 End-stage ischaemic and
dilated CM
12 (inCHF)
5(exclusivetoCHF) 2 (exclusiveto non-CHF)
Idiopathic dilated CM101 Pro-apoptotic genes Tumour necrosisfactor-a
signallingpathway
Idiopathic dilated CM102 Left ventricularassistdevice
(LVAD) support
1,374 genes werereported as ‘increased’and1,629as ‘decreased’
End-stage CHF103 LVADsupport 47 (pre-versus post-LVAD)
End-stage CHF104 LVADsupport Twodistinctgroupsidentifiable withpresent orabsentLVAD
InastudybySteenmanetal.,the expression profiles of four specificpathophysiologicalcardiacsituations wasanalysed: i) left ventricle(LV)from NF heart;ii)LV from F heart affected byDCM; iii)LV from F heartaffected byICM;and iv) right ventricle(RV)fromF heartaffected byDCMor ICM.94Microarrays representing approximately 12,000
humangenes wereutilised.The authorsidentified1,306genes with asimilarexpression profile inallfourcardiacsituations. Theupregulation of embryonicmyosinalkalilightchain-C1 (MLC1emb),calponinand SM22 represented a reversal to developmentalgene expression such as occursincardiac remodelling and CHF. The expression ofsmoothmuscle myosinheavychainandsmoothmusclea-actin represented a dedifferentiation process.Severalapoptosis-related genes were identified inF LV and/orRVsamples,withCDKN1A showingthemost marked activation.Stress-induciblemetal
-lothioneinwasalso upregulated; thisfunctionsasanantioxidant and inhibits theproduction of atrial natriuretic factor.95Novel
genesincluded AF1a, atransmembraneprotein,96and
SH3BGR.Relativelyfewgenes were differentiallyexpressed betweenF LV and F RVsamples, yet themost marked differencewasfound in the geneprofiling, anactin-binding proteinbeing implicated in the control of actin polymerisation and cytoskeletal reorganisation.97Finally, nogenes were
significantlydifferentiallyexpressed betweenDCM and ICM F hearts,which indicates thatall hearts were clinically infailure.
Inaseparatestudy,oligonucleotide arrayshave identified specific genesexpressed in patients with CHF,screeningover 7,000genesin twoNF and twoF humanhearts with diag-noses of end-stage ICM and DCM.98Genes were categorised
intofive groups:(a)cytoskeletaland myofibrillargenes (myomesin,non-sarcomericmyosin regulatory lightchain-2[MLC2] and ss-actin); (b)genes responsible for the degradationand disassembly of myocardial proteins(alpha-1-antichymotrypsin, ubiquitin andgelsolin); (c)genesinvolved in metabolism (ATP synthase alpha-subunit,succinate dehydrogenase flavoprotein [SDH Fp]subunit,aldose reductase and translocator of inner membrane-17 [TIM-17]preprotein translocase); (d)genes responsible for protein synthesis (elongationfactor-2[EF-2], eukaryotic initiation factor-4AIIandtranscriptionfactor homologu e-HBZ17);and (e)genesencodingstress proteins (a-B-crystallin and m-crystallin).The study focusedon thenovel striated muscle LIMprotein-1 (SLIM1)gene asbeing consistently downregulated inCHF.Althoughtheprecise function of SLIM1has notbeendetermined, it ishypothesisedthat it mayactasascaffold for interactionbetween thin fila-mentsandthe cytoskeleton,99inasimilar way to muscle
Lin-11-Isl-1-Mec-3 (LIM) protein (ML). Myosin-like protein (MLP)knockout mice have been shown todevelop DCM.100All ofthe identified genes may contributeto
development of the heartfailurephenotype andrepresent compensatory mechanisms to sustaincardiac functionin failing humanhearts.
Similarly, Steenbergenetal.used DNAmicroarray profiling toinvestigate changesin the expression of genesinvolved in the apoptosis that occurs inhumanidiopathic DCM hearts that haveprogressedto CHF.101Theydocumented altered
gene expressionconsistent with apro-apoptotic shift in the TNF-asignalling pathway.Specifically,theyfound decreased expression ofTNF-a- and NF-kB-induced anti-apoptotic genes such asgrowth arrestand DNA damage-inducible(GADD) 45-b,Flice inhibitory protein(FLIP)andTNF-inducedprotein 3 (A20)genes.Theyalso observed a significant decrease in the phosphorylation of Bd-accelerating apoptosis (BAD)at Ser-112, which isalso consistent with arole forapoptosisin heartfailure.
Oneoption for improving left ventricular functionin CHF is theuseof aleft ventricularassistdevice(LVAD).The unloadingof the F heart with anLVAD can decrease cardiac mass and myocyte size and has the potential to improve contractile function. Several studieshave documented changesingene expressionbrought aboutby the presenceof an LVAD. The effect of chronic ventricular unloading on myocardial gene expression wascompared before and after LVAD support in seven patients with idiopathic DCM and end-stage CHF.102Onaverage,1,374 genes werereported as
‘increased’and 1,629 ‘decreased’ after LVAD support. Upre-gulated genes included a large proportion of transcription factors, genes related to cell growth/apoptosis/DNA repair, cell structure proteins, metabolism and cell signalling/com-munication. LVAD support resulted indownregulation of genescoding forcytokines. Similarly, Chen et al. performed transcriptional profiling using a spotted cDNA microarray with12,814unique clones on pairedsamples of LVobtained before and after placement of anLVAD in 11 patients.103The
most significantly upregulated gene was the G-protein-coupled
receptor APJ,the specific receptor forapelin.Blaxall et al. also examined gene expression, performing oligonucleotide arrays with LV samples from six male patients whichwere harvested during LVAD placement and at the timeof explantation.104 These authors documented a significant
difference in the corresponding LVAD-mediatedregulation of gene expression and were ableto distinguish, in a blind manner, patients with different CHF aetiologies.
Finally,microarrays wereutilisedon paired samples of human tissue from patients with CHFwith and withoutan LVAD.105 After several statisticalanalyses of the data,
In summary, microarrayanalysis of CHF can reveal the underlyingmolecular mechanisms regulating hypertrophyand proliferation, as well asapoptosis of cardiomyocytes. These studies revealedthe dysregulation ofvarious molecular path-waysinvolved inCHF.
E
xpr
e
ss
i
on pro
fi
l
i
n
g
o
f CHD
Congenitalheartdefectsaccountfor thelargest number of birth defectsinhumans.4The application of expression profiling
in patients with CHD is stillinitsinfancy.Todate,only one microarray studyhasdocumented gene expressioninCHD.
Microarrayanalysis wascarriedout incardiacsamples col-lected during cardiacsurgery, from six normal samplesand from55patient samples withvarious cardiac defects.106
HumanUnigene Set-RZPD2 cDNA arrays with12,657 known genes wereused.The study identifiedspecific mol-ecular signaturesfor tetralogy of Fallot (TOF),ventricular septaldefects (VSDs)and right ventricular hypertrophy (RVH), eachof which hasa distinctgene expression profile. TOF and RVHwere foundto be associated with genes of variousfunctionalclasses, and canbe clearlydistinguished by different molecular signatures. The TOFsignature consists of manygenesinvolved in the cellcycle and cardiac development (egupregulation ofswitchnuclear B-siteprotein[SNIP], A2BP1and KIAA1437, and downregulation ofSTK33, BRDG1and TEKT2)andtheupregulation of ribosomal proteinsS6,L37a,S3A,S14and L13A.The RVHsignature includesgenes mainly involved in thestress response, cell proliferationandmetabolism— forexample,theupregulation of ADD2.VSDshave beenassociatedwith aspecificsignature consistingof downregulation of genesfor ribosomal proteins S11,L18A,L36, LP0,L31andMRPS7, and downregulation of genesinvolved incell proliferation, differentiationand apoptosis (egS-adenosyl-methionine decarboxylase [AMD1], receptor interactingprotein serine-threonine kinase-3[RIPK3], Egl-Nine gene family[EGLN1], human siah bindingprotein-1 [SIAHBP1], andArmadilla repeat-proteinin velo-cardiofacial syn -drome [ARVCF]). Severalionchannelgenes, including SLC26A8,SLC16A5,SLC4A7,KCNS2andKCNN3,were found tobe differentiallyexpressed inVSDtissues.The study alsoidentified heartchamber-specific expression ofmany genes.The humanheartconsists oftwoatria andtwo ven-tricles.The atria have beenfoundtohave higherexpression of genesforECMoractin modulation,such asclevage stimu-lating factor-3 (CST3)and procollagenCOOH-terminal proteinase enhancer protein (PCOLCE),translationfactor EEF1Aandthe DNA helicase RecQprotein-like-4(REQL4) and apotassiumchannel subunitKCNIP2.Genes with higher expressionin theventriclesencodeproteins forcytoskeleton contraction, metabolism–energy turnoverand cellcycle– growth, and includeTMP1,humanfactorH-likeprotein(FHL1) andthemuscle ankyrin repeat protein(ANKRD2).
C
on
c
lus
i
ons
The availability of cDNA andoligonucleotidemicroarrays withseveral thousand humangenes makesit possibleto study globalchangesingene expressioninCAD, CHF and CHD. Thesestudieshave identified novelgenesand pathways involved in the generation ofthese diseasesatboththeorgan/ tissuelevel (coronaryarteries, cardiactissues)and cell level (endothelialcells,vascularSMCs,macrophages).Itis import-ant to notethat several studieshave confirmedthe expression of genes previously linkedwiththese diseaseprocesses, suggestingthatexpression profilingwithmicroarraysisavalid approach for identifying gene expressionalterationsassociated with disease.Yet,many morenovelgeneshave alsobeen identified and will serve asavaluableresource foridentifying novel pathwaysinvolved in the generation of disease.Withthe continueduseofthis technology, and anaccelerated appli-cation to these diseases,wewilleventuallyidentifya common group of genes whichwillbe ableto serve as markersfor these diseaseprocesses ona global scale.Itisimportant, however,to notethatdifferent studieshave identified different sets of genes associated withthese diseases. Thus, themicroarray results remain to bereplicated inanindependent set of samplesand caution needs tobetaken when interpretingtheseresults.In addition, amajor limitation ofmicroarrayanalysisis that it is usually difficult todistinguishwhether the identified differ-entialgene expression patternsarethe causeor the conse-quenceofthe disease.Follow-up studies withtransgenic overexpression orknockout ofthe geneof interestinanimal modelsareneededto solvethisissue.Furthermore,microarray analysisisbiasedto the genes on the arraysand cannotbeused toevaluatelow-abundant transcripts. Microarrayanalysisalso resultsinfalse-positives; itis therefore crucial that resultsare confirmedusing conventional technologies,such as quanti-tativereal-time PCR,quantitativereverse-transcriptionPCR, Northern blot, Westernblot orimmunostaining analyses. Finally,the expression level ofmRNA does not necessarily reflect the expression oftheprotein, andthepathogenic mechanism of a diseasemayinvolveprotein modifications such as phosphorylationand glycosylation.Under these cir-cumstances,proteomics will provetobe apowerful, alternative technology.The first proteomicsanalysishasbeen performed forCAD by the authors ofthis paper, andthestudyidentified the ferritin light chainasa significant markerfordiseased coronaryarteries.3Itisimportant to point out that proteomics
cananalyse farfewergenes thancan microarrays, as the res-olution ofthe common two-dimensionalgel separation of proteinextractsis limitedto onlya few thousandprotein spots. Proteomicsalso shares some common limitations with microarrayanalysis;forexample, itcannotbeusedtoevaluate low-abundant proteins/genes.
biologyand biochemistry will validatethese hypothesesand providenovelinsightsinto the pathogenesis of disease.These studies willeventuallygeneratenoveldiagnostic and thera-peuticmarkers, and identify potentialdrug targets whichwill serveto bring about more effectivemanagement of cardio-vasculardisease.
Ack
nowl
edg
m
e
nts
This workwas supported bygrantsfromNIH/NHLBI(HL65630and HL66251)and anAmericanHeartAssociationEstablished Investigatoraward
toQ.W.
Refe
r
e
n
ce
s
1. Gabbing, M.and Weary, G. (2002),‘Anintroduction toDNA chips: Principles,technolo
