Telomere Biology and Thoracic Aortic Aneurysm

16 Pages·2017·1.3 MB·English

International Journal o f
Molecular Sciences
Review
Telomere Biology and Thoracic Aortic Aneurysm
ThomasAschacher1,* ID,OliviaSalameh2 ID,FlorianEnzmann3,BarbaraMessner1
andMichaelBergmann4,5 ID
1 CardiacSurgeryResearchLaboratory,DepartmentofSurgery,MedicalUniversityofVienna,
WaehringerGuertel18-20,1090Vienna,Austria;[email protected]
2 PlasticandReconstructiveSurgery,DepartmentofSurgery,MedicalUniversityofVienna,
WaehringerGuertel18-20,1090Vienna,Austria;[email protected]
3 DepartmentofVascularandEndovascularSurgery,ParacelsiusMedicalUniversity,
MuellnerHauptstrasse48,5020Salzburg,Austria;[email protected]
4 SurgicalResearchLaboratories,DepartmentofSurgery,MedicalUniversityofVienna,
WaehringerGuertel18-20,1090Vienna,Austria;[email protected]
5 ComprehensiveCancerCentre,1090Vienna,Austria
* Correspondence:[email protected];Tel.:[phone]
Received:1October2017;Accepted:19December2017;Published:21December2017
Abstract: Ascendingaorticaneurysmsaremostlyasymptomaticandpresentagreatriskofaortic
dissectionorperforation. Consequently,ascendingaorticaneurysmsareasourceoflethalitywith
increasedage. Biologicalagingresultsinprogressiveattritionoftelomeres,whicharetherepetitive
DNAsequencesattheendofchromosomes. Thesetelomeresplayanimportantroleinprotection
ofgenomicDNAfromend-to-endfusions. Telomeremaintenanceandtelomereattrition-associated
senescenceofendothelialandsmoothmusclecellshavebeenindicatedtobepartofthepathogenesis
of degenerative vascular diseases. This systematic review provides an overview of telomeres,
telomere-associated proteins and telomerase to the formation and progression of aneurysms of
thethoracicascendingaorta. Abetterunderstandingoftelomereregulationinthevascularpathology
mightprovidenewtherapeuticapproaches.Measurementsoftelomerelengthandtelomeraseactivity
couldbepotentialprognosticbiomarkersforincreasedriskofdeathinelderlypatientssufferingfrom
anaorticaneurysm.
Keywords: telomeres;telomeremaintenancemechanism;telomerase;aging;aorticaneurysm
1. Introduction
Telomeres are tandem DNA repeat sequences at the end of chromosomes, which maintain
genomic stability during cell divisions [1–3]. Unfortunately, telomere length (TL) shortens with
everycellreplication. ThisresultsintheDNApolymeraseinabilitytopreciselyreplicatethesetof
chromosomes[4,5]. The“endreplicationproblem”resultsinthecriticalshorteningofTLorinstability
of the telomeric binding proteins (TBPs). This induces an arrest of cell division or a replicative
senescence [6–9], which may cause genomic instability. On average, cells reach senescence after
approximately50divisions. TLcanreflectcellularturnoverandisthereforeusedasasurrogatemarker
forageandasafeasiblebiomarkerforage-relateddiseases. Theseincludecardiovasculardiseases
andinfections,whichareproventohaveacceleratedtelomereshortening[10]. TLhasitspotential
prognosticvaluerelatedtomarkingthespecificstagesofdiseaseprogression. Despitethevalueof
maintainingTL,alimitednumberofcells,especiallyincellswithahighturnover(e.g.,tumorand
epithelial cells) are able to reactivate the enzyme telomerase. Telomerase, a reverse transcriptase
enzyme,addsrepeatsequencestotheendofchromosomes[7,11]. Thisensuresprolongedcellsurvival
andextendedproliferation[3,5]. Moreover,telomerasepresentsextra-telomericfunctionsofenhancing
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Int.J.Mol.Sci. 2018,19,3 2of16
cellrepairmechanismsandstressresistance,aswellasdown-regulationofapoptoticeffectors. Seenas
awhole,itisclearthattelomerasefunctionssupportDNAstability[12–14].
Aortic aneurysms are characterized by local inflammation with degeneration around the
aorta, which leads to weakening and widening of the vessel. The formation of aortic aneurysms
can be congenital or acquired and occur at different locations of the thoracic or abdominal wall.
Abdominalaorticaneurysms(AAA)arestronglyassociatedwitharteriosclerosisandinflammation[15],
whilethoracicaorticaneurysms(TAA)areconsequencesofdegenerativeprocesses,hypertensionor
geneticmutationsinraredisorders(Ehlers-DanlosorLoeys-DietzSyndrome)[16,17]. Thefindings
ofMorgenetal. demonstratethatadvancingageisassociatedwithtelomereuncappinginarteries,
whichislinkedtosenescence,whichoccursindependentlyoftelomereshortening[18]. Whileithas
beenconfirmedthatchronicinflammationinAAAplaysacrucialroleofaneurysmformation,the
TAApathwaystillhastobediscussedandrequiresfurtherresearch.
Vascularaginginvolvessenescenceofendothelialcells(EC)andvascularsmoothmusclecells
(vSMC),whichcanbecausedbytelomereshortening. Severalstudieshaveshownanassociationof
thereductionofTLinbloodcellswithseveralvasculardiseases[19–24]. Amajorvasculardisease
affectingtheaortaisthesporadicthoracicaorticaneurysm,whichhasparticularlyincreasedincidence
intheelderlypopulation[16,25,26].
TheaimofthisreviewistosummarizetheroleofTLandmaintenancebytelomeraseinvascular
diseasesofthethoracicascendingaorta. Wediscussedtheresultsandmainconclusionsthathave
identifiedshorterTLasaprimaryabnormalityinthepathogenesis[27,28]andtheinfluenceoftelomere
functionontheriskofvasculardiseases.
2. Telomeres: Structure,FunctionsandMaintenance
Themainfunctionoftelomeresistoprotecttheendofchromosomesduringreplication. Thisis
essentialforprotectingtheintegrityofgenomicmaterial. Thus,completereplicationoflinearDNA
moleculesisnotpossible,whichwasoriginallyproposedasthe“endreplicationproblem”,byJames
WatsonandAlexeyOlovnikov[29,30]intheearly1970s. TheremovaloftheterminalRNAprimeron
thelaggingstrandleavesanun-replicatedgap,resultinginlossofterminalsequencesandgenomic
instability. Theabilityoftelomerestosafeguardgenomicmaterialisenabledbythedynamicstructure
oftelomeresandTBPs. Humantelomerescontainrepeatsofsequence3(cid:48)-CCCTAA/5(cid:48)-TTAGGGthat
have 2–50 kilobase pairs and a G-tail of 100–250 bases detected throughout the cell cycle [31–33].
However, the “Shelterin complex” contains up to six core components. These are telomere repeat
bindingfactor1and2(TRF1andTRF2),TRF1interactingnuclearfactor2(TIN2),protectoroftelomeres
1(POT1),repressor/activatorprotein1(RAP1)andtripeptityl-peptidase1(TPP1),wherebythreeof
thesefactorsareassociateddirectlywiththetelomericDNAeitherinitssinglestranded(ss)(POT1)
or double stranded (ds) (TRF1, TRF2) form. These components play an essential role in telomere
protectionandtelomeraseregulation[33]. TheShelterincomplexesselectivelybindwithhighaffinity
to areas of the telomere where ss/ds-DNA junctions carry POT1 and TRF1 or TRF2, such as the
3(cid:48)-telomericend[34]. Telomericzincfinger-associatedprotein(TZAP),aspecifictelomere-associated
protein,bindstolongtelomereswithlowconcentrationsofShelterincomplexbycompetingwithTRF1
andTRF2. TheTZAPbindingtriggerstelomeretrimming,whichisanadditionalmechanismofTL
control[35]. However,depletionofTRF2induceschromosomeend-to-endfusions,whichresultin
lossoftelomericsequencesandconsequentlyleadstocriticaltelomereshorteningfoundinhuman
cancerandcells[36].
Telomere-associated proteins influence the control of TL and DNA damage, which involves:
(i)proteinsactingasactivatorsoftheWntsignalingpathway;(ii)modulatingAtaxia-telangiectasia
mutated kinase (ATM)-dependent Ataxia telangiectasia and Rad3-related protein (ATR), which is
activatedinresponsetodamagedcausedbyDNA-doublestrandbreaks(DSB)[37];and(iii)poly-ADP
ribose polymerase-1 (PARP-1), which is important for the normal or abnormal recovery of DNA
damage[38]. Inparticular,TRF1andTRF2canbemodifiedandregulatedbyPARPenzymes[39,40].
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Telomere homeostasis involves different factors that are involved in telomere shortening or
attritionandmaintainingTL.AnewtechniquedevelopedintheMaiLaboratoryinWinnipegallows
the detection of telomere anomalies (attrition, aggregates) in 3D [41,42]. Telomere shortening is
posed by telomere attrition during cell division or direct DNA damage on telomeric sequences.
TelomericDNAdamagecanbecausedbyoxidativestress,lossordeficiencyofTSBordegradationof
RNAprimersofproteinsinvolvedinDNArepair(e.g.,Ku70/80,PARP-1). Inaddition,homeostasis
oftelomeresisregulatedbyDNAmethylation,whichenablestelomeric-stabilizedproteininhibition
orexpression[41].
Telomerase counteracts telomere shortening to maintain telomeres in the vasculature.
Still,telomereattritionoccurswitheachmitoticcycleduetothefactthattelomeraseactivity(TA)is
typicallyloworabsentinhumansomaticcells[3,42,43]. Telomerasecontainstwomaincomponentsto
compensatethislossbyaddingDNAsequencestotheendofchromosomes. Thefirstcomponentis
hTERC,anRNAcomponent,andthesecondhTERT,areversetranscriptase. WhilehTERCexpression
extendstelomerasereactivation,hTERTlimitstelomeraseactivity[44–47]. Further,hTERTregulates
istranscriptionalgenesinvolvedincellproliferationandextensionofthelifespaninnumerouscell
types,suchasvSMCs,fibroblasts(FBs)andECs.
Thelatestfindingsofthenon-canonicalfunctionsoftelomerasehaveshownthathTERTexpression
influencedcellgrowth,regardlessoftelomeremaintenance[48]. Moreover,extra-telomericfeatures
oftelomeraseuncoveredanindependentroleoftelomereextension[49]. Indetail,thenon-canonical
functionsofthehTERTcatalyticsubunitareparticularlyinvolvedincancerprogressionwithtwomajor
signalingmechanisms,whicharenamelytheNF-κBandWnt/β-cateninpathways. TERTconducts
asatranscriptionalmodulatorintumorcellstosustainitsownlevelsandtocontroltheinductionof
targetgenescriticalfortumorcellsurvivalandproliferation[49].
Asecondandraremaintainingmechanismoftelomerelengthfoundinapproximately10–15%of
tumorsisarecombinantprocess,thealternativelengtheningoftelomeres(ALT),whichresultsfrom
homologousrecombination[50,51]. TheALTmechanismhasnotbeenfoundinhumanaorticcellsand
hasnotbeenshowntobeactiveinTAAorAAA.
Telomere exhaustion is a major age-dependent problem as aging is known to be a risk factor
forvasculardiseases[48]. Theenvironmentalimpactmightalsobeofgreatimportancefortelomere
shorteningduringthedevelopmentofaorticdiseases. Thus,formanyreasons,TLandtelomeraseplay
acriticalroleinaneurysmformation,astheyareassociatedwithadverselifestyleandcardiovascular
diseases(CVD).
3. TelomeresandPathobiologyofAorticAneurysms
Telomereshavealengthgeneticallydeterminedatbirth. TLprovidesapotentialmarkerforan
individual’sbiologicalage. Thereisalackofknowledgeaboutgeneticpredispositionsintelomere
maintenanceduringaginganditsdirectrelationtotheintegrityoftheaortictissue. Thereislittle
evidence for an association between the genomic determinants of TL and the risk of AAA [52].
Balistreriandhisgroupshowedacorrelationbetweenage-dependentaneuploidyandTLofthehuman
vascularendothelium[53]. TLwasinverselycorrelatedwithageandfurtherincreasedsignificantly
withthefrequencyofaneuploidyofvascularECs.
Telomere shorting and the associated genetic instability in vasculature plays a key role in
TAA etiology [54]. Recent studies can be divided into two groups: (a) measurements of TL and
telomeremaintenancemechanismsindiseasedaortictissue,comparedtohealthyaortictissue;and
(b)comparativestudiesofwhitebloodcells(WBC)TLwithhealthyanddiseasedaortictissues. Inthis
section,thetelomericspecificityofaortictissuewillbesummarized.
Artery walls of large vessels consist of layers known as the tunica intima, the tunica media
and the tunica adventitia. Their main cell types include ECs, vSMCs, FBs and macrophages [55].
TheintimallayerisessentiallycomprisedofECs. ThesecellshavesubstantialinfluenceonvSMC
differentiation [56] and recent studies have showed that ECs can directly influence the vSMC
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phenotype[57,58]. ThebloodvesseltoneismaintainedbydifferentiatedvSMCs,whichregulatethe
bloodpressurethroughconstrictionorrelaxation. ECsandvSMCsarekeyplayersinvasculogenesis,
whichisimportantforstructuralstabilityandmaintenanceduringcellularstressorvasculardamage
topreserveaorticintegrity.
Previously, Okuda et al. assumed that a higher rate of telomere attrition enhances the rate
of senescence of endothelial cells, which further increases the predilection for vascular disease.
Cellsenescencehashighersignificanceintheirmainfocus,thedistalaorta. Theyalsoanalyzedthe
proximalaortictissuewiththefollowingmainfindings: theTLofintimaltissuewasage-dependent,
whereas the proximal medial aortic tissue near to the ascending aorta showed no significant
age-dependenttelomereattrition. Localfactorssuchasshearwallstressmayinfluencesuchfindings
beingpartiallyrepresentativetovascularcellsenescence[59].
Theassociationsbetweentelomerase,aorticaneurysms,epidemiologicalandclinicalvariables
have been investigated in different studies, although they were limited to AAA. Dimitroulis et al.
suggested a protective role of telomerase against AAA formation as studies showed that patients
suffering from AAA had attenuated endothelial telomerase expression compared to controls [60].
However, there are almost no studies on this subject, which is why we designed a study to
evaluate TL in TAAs, which involves the use of bicuspid (BAV) and tricuspid (TAV) aortic valves
in TAAs [61]. We isolated aortic SMCs and subjected them to cell biological and gene expression
analyses. TheobtaineddataindicatethataneurysmalSMCshavereducedproliferationandmigration
ratescomparedtocontrols. Moreover,wedemonstratedshortertelomeresinaneurysmscompared
tocontrols. FurtheranalysisshowedadifferencebetweenSMCsisolatedfromTAAscomparedto
thoseisolatedfromBAVandTAV.BAVSMCshadsignificantlyshortertelomeres,whereasTAVSMCs
showed reduced metabolic activity. Our study provides evidence that TAA-associated aortic wall
disintegrationshowssimilaritiesinbothBAVandTAV,althoughtherearestillsignificantdifferences.
Severalpreclinicalstudiessupporttheideathatage-relatedendothelialandmyocytedysfunction
couldcontributetothedevelopmentofAAA.Bhayadiaetal.investigatedaortasintelomerase-deficient
miceandfoundsignificantlyhigherexpressionsofstress-inducedsenescencemarkersp16(INK4a)
andp19(ARF)comparedtowild-typemice. Thisobservationeliminatedthepossibilitythatlossof
telomeraseexpressionorlossofpreviouslyreportedtelomere-independent,non-canonicalfunctions
oftelomerase[62,63]resultinendothelialdysfunctionoftheaorta. FocusingonECs,Bianchessiet
al. aimedtodefinewhethermitochondrialDNA(mtDNA)-transcribedlong-non-coding-RNAplays
aroleinvascularaging. SenescentcellsinaortasofoldmiceshowedincreasedlncRNAexpression.
TransientoverexpressionoflncRNAwasshowntoinduceG2/Mcellaccumulationandreplicative
senescence in ECs. Invitro analysis established that these changes are induced in ECs, but not
in vSMCs [64]. Boe et al. established plasminogen activator inhibitor-1 (PAI-1) as an important
determinantofvascularsenescenceinvivo,whichisoneofthemajoranti-fibrinolyticproteins. Itis
expressedinECsandappearstoplayapivotalroleinvascularagingandhypertension. Telomeresand
TLhavebeenexaminedinaortictissuefromanimalstreatedwithPAI-1.Theresultsshowedsignificant
inductionofsenescenceandacceleratedaging,leadingtotheconclusionthatPAI-1inhibitionmaintains
TL[65]. Additionally,Thannickaletal. describedthattheremovalofsenescentcellscanprotectthe
organismagainstaging[66].
Insummary,shorteningoftelomeres,reducedtelomerasefunctionandcellularsenescenceof
vSMCsandECsseemstoplaycrucialrolesinthedevelopmentofTAAs. Furtherstudieswithagreater
cohortofsamplesarenecessarytounderstandtheroleofgenetically-determinedaorticTL.
4. RegulationofTelomeresandSenescenceinCellsoftheAorticWall
Given the high impact of TL in cells of the aortic wall, it is essential to understand telomere
regulationinanage-relatedmanner. Previousinvivodatahavesuggestedthattelomeraseactivation
ispivotalfortheregulationofvSMC,ECproliferationandlong-termcellviability[67].
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The age-dependent function of EC impairs angiogenesis and the development of vascular
diseases[68,69]. InvivoobservedECisremarkablyinertuntilactivatedtoproliferateaftertraumatic
injuries,inflammationandtumorformation[70].
Telomere attrition has been detected in senescent ECs, even though telomerase in these cells
inhibitstheonsetofsenescence. Telomereshorteningcorrelateswithreducedgrowthrateofolder
humanECs[71,72]. TelomeraseectopicoverexpressionincreasestheproliferationrateofhumanECs
andvSMCs[73,74].Studiesdemonstratethatthereactivationoftelomeraseintelomerase-negativecells
canbeinducedbyhTERT.ContrarystudiesinmicelackingTAshowcompromisedcellproliferation
andreducedtumorigenesis. Indetail,phosphorylationofTERTisimportanttoactivatetelomerasein
thenucleusduringvSMCproliferation[75]. Yangetal. describedaresistanceofhTERT-expressing
ECstotheinductionofapoptosisbydifferentconditionsrelatedtoparentalcellsatsenescence[76].
Moreover,thisgroupreportedaninverserelationshipbetweenTAandapoptosisinthesecellsforthe
firsttime. Thetelomereswereonlyelongatedtoaspecificlength,whichallowedthecellstobypass
senescence,althoughtheydidnotreachTLasdescribedforhTERT-immortalizedcells.
AnotherstimulusforTAinECsisthefibroblastgrowthfactor2(FGF-2). FGF-2expressionalso
delaystheonsetofsenescenceinaorticECs[77]. However,sirtuin-6(SIRT6)ishighlyexpressedin
ECs,fibroblasts,embryonicstemcellsandtumorcelllines,whereitprotectsDNArepairandtelomere
maintenance[78–80]. Inthiscontext,thefindingthatadepletionofSIRT6inECsinducesasenescent
phenotypesuggeststhatincreasingthelevelsoractivityofthisproteinmaybearelevantapproachfor
delayingvascularageing.
Importantly, senescence of vSMCs contributes to aging and age-related diseases of the
cardiovascularsystem. ProlongedcellarrestinmitosismaycausedisorderofTRF2andthetelomere
structure[81]. Accordingly,Bielak-Zmijewskaetal. analyzedthepathwaysofreplicativesenescence
ofvSMCsinvitro. Thecellularsenescencewasinducedindoxorubicin(DOX)-treatedcardiomyocytes,
whichledtoastress-inducedprogramofsenescence(SIPS)inthesecellsaspreviouslydescribed[82].
Amajorroleoftelomeredysfunctionisthestress-inducedsenescenceandapoptosis[83]. Ogawaetal.
demonstratedthatmitogen-inducedTAinvSMCsisinhibitedbyperoxisomeproliferatoractivated
receptor-γ(PPARγ)ligands.Furthermore,vSMCtreatedwithPPARγligandsreducedcellproliferation,
whichwaspreventedincellsoverexpressingTERT.ThisfindingcharacterizesTAasanimportant
anti-proliferativetargetforPPARγligands[84].
Hypoxia is a well-known regulator of vascular function and structure, which extends the
proliferation capacity of vSMCs through increased TA. Conversely, vSMC growth arrest occurs
aftertelomeraseinhibition[85],whilesenescentvSMCsareacceleratedbyoxidativestress-induced
DNA damage, inhibition of telomerase and marked TL in arteriosclerosis [86]. Similar findings
are found in ECs, where oxidative stress induces a downregulation of telomerase and increases
telomeredamage[87]. AntioxidantspreventedTERTdownregulationinECs[88]. Inatherosclerotic
vessels,telomereshorteningispronetohighhemodynamicstresses,whichmightalsoenhanceEC
turnover[59,71].
5. FactorsAffectingTelomeresandtheRiskforTAAFormation
Invitro and invivo findings have analyzed the link between telomeric integrity and factors
influencingTAAformation. First,wediscussunifyingpathophysiologicalmechanisms,whichare
responsibleforageingandage-relateddisorders.
5.1. OxidativeStress
Accumulationofoxidativedamageplaysanimportantroleinage-relatedtelomeredysfunction
ofvascularcells[89]. Oxidativestressisdefinedasanincreaseintheintra-cellularconcentrationof
reactiveoxygenspecies(ROS)[90,91]. ROSaregeneratedduringtheregularmitochondrialelectron
transportchain. OtherROSspeciescanbederivedfromsuperoxideandhydrogenperoxide,whichare
associatedwiththedepletionofextracellularmatrixproteins. ROSisproposedtobeinvolvedinTAA
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development. Inaortictissue,increasedROSlevelsweredetectedinaneurysmalaortascomparedto
non-aneurysmalaortasofpatientsandwereconfirmedtoplayanimportantfactorinthedevelopment
ofaneurysms[92]. KnockoutmicewithsuppressedROSproductionhadreducedTAAformation[93].
Moreover,ROSinducesgenomicinstabilityandlossofDNAstabilizationfactors,whichwasshownin
thoracicaneurysmalvSMCs[94].
Telomerase is not only located within the nucleus and cytoplasm, but also be found in the
mitochondria[95]. Thetelomeraselocatedinthenucleusplaysacrucialroleintelomereelongation,
geneexpressionregulation,chromatinorganizationandDNA-damageresponses[96]. Incontrast,the
telomerasefoundinmitochondriaisinvolvedinregulationandinfluencesapoptosis,stressprotection,
transfer-RNA-dependentreversetranscriptaseandRNA-dependentRNApolymerase. However,in
context with ROS and oxidative stress, mitochondrial telomerase reduces the production of ROS
andprotectsmitochondrialDNAfromdamage[62,63]. Interestingly,telomeraseseemstohavean
anti-apoptoticrole,withthepotentialtoblockmitochondrial[14]anddeathreceptorpathways[97].
TelomereshorteninghasbeenassociatedwithoxidativestressbyfindingoxygenradicalsinEC,
vSMC,epidermalcellsandWBCsfromAAApatients[98]. Mostrecently,Rehhetal. demonstrated
thattripletguaninespresentintelomericTTAGGG-repeatsislinkedtothepreferentialaccumulation
ofoxidativestress-relateddamageintelomeres[99]. AninversecorrelationwasdetectedbetweenTL
andhighlevelsofoxidativeDNAdamageinWBCsfromAAApatients. Thissuggeststhatoxidative
stresshassystematiceffects,similartotelomereshortening[97]. Therelationshipbetweentelomere
shorteningandoxidativestressisstillunderdiscussion.
ThestudiesaresuggestingtheideathatoxidativestressinhibitsTAandcausestelomereattrition.
Moreover, the non-canonical mitochondrial specific role of TERT is focused on protection against
oxidativestress. Therefore, aregulationofTERTwillbeapromisingtargetinthedevelopmentof
therapeutictreatments,whichaffectingtelomerase,inagingandtelomererelateddiseases.
Beyeretal. identifiedthatincreasingTAreversesthepathologicalphenotypeseeninCVD[100].
ThisstudysupportsthehypothesisthatTAinmitochondriaregulatesROSproductionwithdirect
physiologicalimpact. Moreover,newfindingsinCVDshowedthatnicotinamideadeninedinucleotide
phosphate(NADPH)oxidaseplaysanessentialroleinthedevelopmentofdiseases. Forexample,
Xu et al. have demonstrated that NADPH oxidase increases the proportion of SMC phenotypes
associated with atherosclerotic plaques vulnerability [101]. There is a lack of data related to these
findingsinTAAs,althoughthisshouldbeapromisingfieldofdiseaseresearch.
5.2. Nutrition
Itiswellknownthatanincreaseofcaloricintakeand,consequently,obesityareknowntoreduce
the average age. Specially, the adipose tissue plays a crucial role, which influences the function
andstructuralintegrityofthecardiovascularsystemasasourceofROS[102]. Unhealthydiet,high
glucose (e.g., fructose) and low-density lipoprotein intake [103] are associated with an increased
risk of CVD. As human primary cardiovascular cells show phenotypic and molecular changes.
Simulated unhealthy diets showed enhanced proliferation of SMCs and increased senescence as
wellaslossofendothelialnitricoxidesynthase(eNOS)inECsinvitro. Further,theproteinrestriction
duringlactationformaternalanimalsshowedareductionofDNAdamageandshortenstelomeres
inaortictissue. Theseresultsareassociatedwithreduced8-hydroxy-2-deoxyguanosine,amarkerof
oxidativestress,whichhasapositiveeffectonchildgrowthandcardiovasculardiseases[104].
5.3. Hypertension
Hypertensionisasignificantcontributortotelomereattrition[105]. Thepotentialrelationship
between TL and human hypertension were examined in several studies. Two large studies, the
Framingham heart study and the cardiovascular health study, have suggested that hemodynamic
stressinlinkedtotelomereshortening[24,106]. IntheFraminghamHeartStudy,leukocytesTLof
hypertensivemalesubjectsaresignificantshortercomparedwiththeirnormotensivepeers.
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Themolecularbasisoftheabnormalityinhypertensionhasnotbeenfullydefinedasvascular
remodelinginarterialvesselswithhighbloodpressure. Studiesintelomerase-deficientmicehave
shownadirectassociationbetweenhypertensionandtelomereattrition[107]. Caoetal. designedan
animalstudywithspontaneouslyhypertensiveratsandanalyzedaortictissue[67]. Telomerasewas
selectivelyactivated,whiletelomereswerelengthenedinvivoandinvitro. Moreover,telomerasewas
foundtoplayasignificantroleinaortictissue.Thus,thedownregulationofhTERTarreststheincreased
proliferationofvSMCsinhypertensiverats. Inaddition,thestudyshowedthatthep53checkpoint
appearstobecrucialforincreasedvSMCgrowthrate.Themoleculartargetingofthisincreasedactivity
maythusprovideawindowofopportunityforpreventinghighbloodpressure. Incontrast,thestudy
fromDimitroulisshowedthatdifferencesintelomeraseRNAexpressionbetweenAAAsandcontrols
wereindependentofotherfactors,suchasageandhypertension[60]. Summarizing,areductionofTL
isassociatedwithincreasedECgrowthratethatseemstobeaconsequenceofvascularshearstress.
5.4. DiabetesMellitus
Diabetic patients are at higher risk of vascular diseases [108] as post-prandial hyperglycemia
increasescardiovascularrisk.Diabetesandhyperhomocysteinemiahavebeenshowntobeindependent
riskfactorsforincreasedprogressionofCVD,includingTAAandAAA[109].vSMCculturesexposedto
highlevelsofglucoseor/andhomocyteine(Hcy)showedincreasedTA,whichresultedinaproliferative
responseofvSMCs[109].Incontrast,Matsuietal.showedthathighplasmaconcentrationsofinsulin
therapymightaccelerateendothelialsenescence[110].HumanECsexposedtohighglucoseincreased
senescencemarkersanddecreasedTA.PreservedTLanddelayedsenescenceinECsunderhigh-glucose
conditionswerepreservedwithnormalconcentrationsofinsulin.Moreover,theseeffectswereassociated
withreducedreactiveoxygenspeciesandincreasednitricoxide(NO).Theseresultsmayhelptoexplain
thecomplicatedrolesofinsulininCVDintheelderly[110].Nevertheless,recentstudieshaveclaimedthat
diabeteshasaprotectiveroleregardingtheformationofabdominalaorticaneurysms.Furtherstudies
needtoelucidatewhetherthisprotectionisbasedontheeffectofdifferentanti-diabeticmedications
(whichareknowntopotentiallyreduceoxidativestresswithinthevascularwall).
6. TelomeresasMarkersinLeukocytes
Intheearly1990s[111],itwasspeculatedthatTLmeasuredinleukocytesisamarkerforincreasing
age.BiologicalageseemstobeamoredescriptivepredictorofCVDratherthanchronologicalage[112].
Non-functional vascular ECs provoke a transendothelial migration and adhesion of circulating
leukocytes. These circulating blood leukocytes reflect the biological age of vascular walls and are
associatedwithareductionofTLandCVD[113,114].
PatientswithAAAshowedshorterleukocyteTLcomparedtocontrols[115]. Themeasurement
of leukocyte DNA mirroring for vascular telomere content seems to be an accurate surrogate for
human vascular age [114]. Additional factors (e.g., genetic and environmental) are involved in
telomereshortening,suggestedbyaninversecorrelationbetweenTLandoxidativeDNAdamagein
awidespectrumofinflammatoryanddegenerativedisorders[98]. Thepathologicalprogressionof
atherosclerosisinAAAisinducedbyaninflammatoryprocess,whichismainlyresponsibleforahigh
cellularturnoverandtherefore,rapidtelomereshortening[116].
The risk factors for telomere attrition and a decrease of TA in TAA patients are significantly
associated with an increased systemic inflammation, age, gender, nicotine abuse and high blood
pressure [117,118]. An inverse correlation between short TL and chronic vascular diseases in a
high-riskpopulationofchronickidneydisease(CKD)patientswasfound[119]. Whereby,themost
significantassociationswerefoundforcarotidarteriesinterventions,TAAandAAA.
Interestingly, the group of Salonurmi analyzed TL in TAAs and demonstrated opposite
results[113]. Salonurmietal. detectedTLbyusingtheCawthon’squantitative(q)PCRmethods,while
othergroupsmeasuredTLbycomparingthemeanterminalrestrictionfragments(TRF)lengthby
usingthemorepreciseSouthernBlotanalysis. Inallstudies,severallimitationsneedtobeconsidered.
Int.J.Mol.Sci. 2018,19,3 8of16
TLvariesbetweendifferentchromosomesandcelltypes. MeasurementofthemeanTRFlengthis
inexacttoanalyzecellulartelomereattrition. Ascriticaltelomereshorteningofasinglechromosome
doesnotaffectingthemeanTRFlength. Therefore,onepointofinterestshouldbethesignalsfrom
telomericDNAdamageinaortictissueandWBCsofsamepatients.
Yanetal. hasdemonstratedsignificantlyshorterleukocyteTLinaChinesepopulationaffected
byaorticdissectionscomparedtoacontrolgroupwithoutvasculardiseases[20]. Afteradjustments
forotherriskfactorstheshortleukocyteTLwasassociatedwithaorticdissection.
Several factors have contributed to the lag of telomere measurements in patients and
data interpretation. This includes technical obstacles for large-scale measurements and lack of
standardizationacrosstechniquesandlaboratories,apartfromthestrongassociationwithriskfactors
inpatients. Inclinicalpractice,age,genderandlifestylealsoplayanimportantrole,whichmakesthe
usageofTLmeasurementasabiomarkerdifficultandcontroversialdiscussed.
7. MechanismtoPreserveTelomereLength
A comprehensive health concept, including maintenance of high-intensity physical exercise
and a well-balanced diet, has been suggested for preserving TL by activating telomerase even in
oldage[120,121]. ThecardiovascularbenefitsofchronicexercisearefoundinWBCsisolatedfrom
enduranceathletes. Comparedtountrainedindividuals,enduranceathleteshaveincreasedTA,higher
levelsofexpressionoftelomere-stabilizingproteinsandsignificantlowerlevelsofcellulargrowth
arrestandcelldeathmarkers. Althoughallparameters,suchasheartrate,bloodpressure,bodymass
indexandlipidprofile,arebeneficialinathletes,thepositiveTBPregulationwasindependent.
In the context of a healthy diet, several nutritional components have been shown to have a
positivecorrelationwithincreasedTA[122]. Balancedintakeofmarineomega-3fattyacidlowersthe
rateoftelomereshortening[123]. Resveratrolfoundingrapes,blueberry,raspberryandmulberry
activateshumannicotinamidephosphoribosyltransferase,sirtuin-4andthereversetranscriptaseof
telomeraseinhumanaorticSMCs. Thisalsodecreasedtheexpressionofp53by50%andincreased
TL,whichdelayedvascularaging[124]. Adietarysupplement, ShoushenGranule, wasshownto
improveatheroscleroticlesionsofthearterialwallbydecreasingtotalcholesterolandlow-density
lipidcholesterollevelsandincreasingTAandTLinperipheralleukocytesandvascularcells[125].
Furthermore,ricebran,aby-productofthericemillingprocess,containsγ-oryzanol,tocopherolsand
phytosterols. Positivelyeffectscanbeattributedtoricebran,aslipid-loweringandanti-inflammatory
effects. Theenzymaticextractofricebranpreventstelomereshorteninginaortaandmononuclear
cellsandreducesvascularapoptosisandatherogenesisinmice. Presently,theexactmechanismby
whichresveratrol,ShoushenGranuleorricebraninducestelomeraseremainsunknown;thesefindings
suggestabeneficialeffectinanti-agingprocessesindiseasedcardiovascularcells.
TLcanalsobepreservedbypharmaceuticals. Aspirin,angiotensin-converting-enzyme(ACE)
inhibitors and statin therapy have been shown to have an anti-senescence effect on the vascular
endothelium[126–130]. Pioglitazone,adrugofthethiazolidinedioneclass,increaseTAinaorticcells
andpreventsstress-inducedcelldeathofECs. Itreduceslevelsofsenescencemarkersp16,cell-cycle
checkpoint kinase 2 and p53 [84,131]. Activation of liver X receptors (LXRs), which are nuclear
receptors,aretriggersforprotectionofarteriesagainstatherosclerosis. LXRligandsstabilizelowlevels
ofsenescence-associatedβ-galactivity,andreversethedecreaseoftelomeraseexpressioninhuman
ECs. ThiseffectofLXRactivationwasassociatedwithreducedROSandincreasedeNOSactivity[132].
8. Conclusions
Geneticdispositions,suchastheEhlers-DanlosSyndromeorMarfanSyndrome,andvascular
riskfactors(e.g.,smokingandobesity)appeartobestrongriskfactorsforAAA.Studiesinanimal
and human trials associate a reduced TL and an inherent TA with DNA damage and increased
predispositiontoformAAAs. Insummary,followingfindingsshowthelinkbetweentelomerebiology
and TAA (Figure 1). (1) Shortened telomeres reduced telomerase function. Therefore, premature
Int.J.Mol.Sci. 2018,19,3 9of16
senescence of vSMCs and ECs are strongly associated with the development of aortic aneurysms;
(2) Aneurysm formation results from abnormal proliferation and/or degeneration of vSMCs and
Int. J. Mol. Sci. 2018, 19, 3 9 of 16
ECsintheaorticwall,whereastheinductionofTAisessentialfortheregulationofvSMCandECs
proliferation,whichoccursindependentlyofprogressivetelomereshortening;(3)Shortenedtelomeres
(3) Shortened telomeres in aneurysmal aortic tissue were correlated with telomere attrition in white
inbalnoeoudr yceslmlsa. laortictissuewerecorrelatedwithtelomereattritioninwhitebloodcells.

Figure 1. Multiple factors are associated with thoracic aortic aneurysm formation. The influence of
Figure 1. Multiple factors are associated with thoracic aortic aneurysm formation. The influence
telomerase activity on vascular smooth muscle cells (vSMC) and endothelial cells (EC) and the
oftelomeraseactivityonvascularsmoothmusclecells(vSMC)andendothelialcells(EC)andthe
regulatory interaction between both cells is of particular importance.
regulatoryinteractionbetweenbothcellsisofparticularimportance.
A considerable number of reports suggest that telomerase and its key components, TERT and
TEARCc,o hnasvide efruanbclteionnus mbebyeornodf trheepiro arbtsilsituyg tgoe lsetntghthatente tleolmomerearesse. aTnAd isit fsokuenyd cino ma pwoidnee nratsn,gTeE oRf Tcealln d
TEtRyCpe,sh aanvde tfiusnsuceti oton bseb ienyvoonlvdetdh ienir aa vbailriiteytyt oofl eanctgivthiteienst, eelsopmeceiraellsy. wTAithis nfoonu-ncadnionnaicawl ifduencrtainongse tohfact ell
typheasvae nndotti sbseueen tofublley inchvaorlavcetderiinzeadv. aInri eatoyrotifc accetlilvsi, titeeslo,mesepreacsiea lslyeewmist hton owno-crkans oansi caa ltrfaunnscctriiopntisotnh at
havcoe-nfaocttobre etnhaftu lrleygcuhlaarteasc ttehreiz eedx.pIrnesasoiornti cocfe lgles,nteesl oimnveorlavseeds eienm tsheto rwegourlkastiaosna otfr acneslcl rpiprotiloifnercaot-ifoanc,t or
thadtifrfeegreunlatitaetsiotnh eanedxp arpeospsitoonsiso. f genesinvolvedintheregulationofcellproliferation,differentiation
andapoTpLt osesiesm. s to be an attractive new biomarker for biological age or for follow-up of aneurysm
proTgLresseseiomns otvoerb yeeaanrs.a Httroawcetivveer,n TeLw nebeiodms taor bkee rinftoerrpbrieotleodg wiciatlh acgaeutoiornf odruefo tlolo twhe- ufoprmofataionne uarnyds m
prothger epsrsoiognreossvioern yoef aTrAs.AH oovwere dveecr,aTdLes.n Leietdtlse tios kbneoiwntne rapbroeutte dtelwomithercea autttrioitniodn uine tthoet heaerflioerrm staatgioesn oafn d
disease. Interestingly, telomere shortening is accelerated prior to the onset of clinical disease.
theprogressionofTAAoverdecades. Littleisknownabouttelomereattritionintheearlierstagesof
A variety of risk factors, such as age, gender, smoking and obesity, have a great influence on both TL
disease. Interestingly,telomereshorteningisacceleratedpriortotheonsetofclinicaldisease. Avariety
and the health of most patients. Therefore, the non-specific modulations of TL suggest a poor
ofriskfactors,suchasage,gender,smokingandobesity,haveagreatinfluenceonbothTLandthe
specificity and limited potential as a biomarker of a specific disease.
healthofmostpatients. Therefore,thenon-specificmodulationsofTLsuggestapoorspecificityand
Hence, the main questions related to telomere regulation and the development of aortic
limitedpotentialasabiomarkerofaspecificdisease.
aneurysms remain unanswered: Does telomere-shortening lead to the development of TAA or does
Hence, the main questions related to telomere regulation and the development of aortic
the DNA damage caused by external factors (e.g., hypertonic blood flow) reduce TL in aortic cells?
aneurysmsremainunanswered: Doestelomere-shorteningleadtothedevelopmentofTAAordoes
Prospective longitudinal studies would be required for accurate delineation of true changes in TL.
theDNAdamagecausedbyexternalfactors(e.g.,hypertonicbloodflow)reduceTLinaorticcells?
Conflicts of Interest: The authors declare no conflicts of interest.
ProspectivelongitudinalstudieswouldberequiredforaccuratedelineationoftruechangesinTL.
Funding: Supported in part by grants from the Medical-Scientific Fund of the Mayor of Vienna (Project-No.
Co1n5fl1i5c5t)s. o fInterest:Theauthorsdeclarenoconflictsofinterest.
Fun ding:Supportedinpartbygrantsf romtheMedical-ScientificFundoftheMayorofVienna(Project-No.15155).
Int.J.Mol.Sci. 2018,19,3 10of16
Abbreviations
AAA Abdominalaorticaneurysm
ALT Alternativelengtheningoftelomeres
BAV Bicuspidaorticvalve
CKD Chronickidneydisease
CVD Cardiovasculardisease
DOX Doxorubicin
ds/ss Double-stranded/single-stranded
DSB Doublestrandbreak
EC Endothelialcell
eNOS Endothelialnitrateoxidesynthase
FB Fibroblast
LXR LiverXreceptor
NAMPT Nicotinamidephosphoribosyltransferase
NO Nitrideoxide
PAI-1 Plasminogenactivatorinhibitor-1
PARP poly-ADPribosepolymerase
POT1 Protectionoftelomeres1
RAP1 Repressoractivatorprotein-1
ROS Reactiveoxygenspecies
RTL Relativetelomerelength
SIPS Stress-inducedsenescenceprogram
(v)SMC (vascular)smoothmusclecell
TA Telomeraseactivity
TAA Thoracicaorticaneurysm
TAV Tricuspidaorticvalve
TBP Telomerebindingprotein
(h)TERC/(h)TR (human)telomeraseRNA-component
(h)TERT (human)telomerasereversetranscriptase
TIN2 TRF1-interactingprotein2
TL Telomerelength
TPP1 Tripeptidylpeptidase1
TRF Telomererepeat-bindingfactor
TZAP Telomericzincfinger-associatedprotein
WBC Whitebloodcell
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