NF-B-Mediated Inflammation in the Pathogenesis of Intracranial Aneurysm and Subarachnoid ...

20 Pages·2017·5.93 MB·English

International Journal o f
Molecular Sciences
Review
NF-κB-Mediated Inflammation in the Pathogenesis
of Intracranial Aneurysm and Subarachnoid
Hemorrhage. Does Autophagy Play a Role?
ElzbietaPawlowska1,JoannaSzczepanska2,KarolWisniewski3,PaulinaTokarz4,
DariuszJ.Jaskólski3andJanuszBlasiak4,* ID
1 DepartmentofOrthodontics,MedicalUniversityofLodz,92-216Lodz,Poland;
[email protected]
2 DepartmentofPediatricDentistry,MedicalUniversityofLodz,92-216Lodz,Poland;
[email protected]
3 DepartmentofNeurosurgeryandNeurooncology,MedicalUniversityofLodz,BarlickiUniversityHospital,
Kopcinskiego22,90-153Lodz,Poland;[email protected](K.W.);[email protected](D.J.J.)
4 DepartmentofMolecularGenetics,FacultyofBiologyandEnvironmentalProtection,UniversityofLodz,
90-236Lodz,Poland;[email protected]
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:29March2018;Accepted:17April2018;Published:19April2018
Abstract:Theruptureofsaccularintracranialaneurysms(IA)isthecommonestcauseofnon-traumatic
subarachnoid hemorrhage (SAH)—the most serious form of stroke with a high mortality rate.
Aneurysmwallsareusuallycharacterizedbyanactiveinflammatoryresponse,andNF-κB(nuclearfactor
kappa-light-chain-enhancerofactivatedBcells)hasbeenidentifiedasthemaintranscriptionfactor
regulating the induction of inflammation-related genes in IA lesions. This transcription factor has
also been related to IA rupture and resulting SAH. We and others have shown that autophagy
interacts with inflammation in many diseases, but there is no information of such interplay in IA.
Moreover,NF-κB,whichisapivotalfactorcontrollinginflammation,isregulatedbyautophagy-related
proteins,andautophagyisregulatedbyNF-κBsignaling. Itwasalsoshownthatautophagymediates
thenormalfunctioningofvessels,soitsdisturbancecanbeassociatedwithvessel-relateddisorders.
Earlybraininjury,delayedbraininjury,andassociatedcerebralvasospasmareamongthemostserious
consequencesofIAruptureandareassociatedwithimpairedfunctionoftheautophagy–lysosomal
system. Further studies on the role of the interplay between autophagy and NF-κB-mediated
inflammationinIAcanhelptobetterunderstandIApathogenesisandtoidentifyIApatientswithan
increasedSAHrisk.
Keywords: intracranial aneurysm; subarachnoid hemorrhage; inflammation; NF-κB; autophagy;
earlybraininjury;delayedbraininjury
1. Introduction
On average, one in 25 persons can be at risk of intracranial aneurysm (IA), a cerebrovascular
disorderwhichmayleadtodevastatingsubarachnoidhemorrhage(SAH)whenrupturesoccur[1].
Inturn,halfofSAHcasesresultindeathinafewweeksafteritsoccurrence,andlessthanhalfofthe
remainingpatientsisabletoconductanormallife.However,thepathogenesisofIAandtheevents
precedingitsrupturearepoorlyknown.Therefore,efficientIApreventionanddetectioncandecrease
the number of life-threatening cases of IA-related SAH. Neurosurgical clipping and endovascular
coilingtreatmentofIAtopreventitsruptureareinvasiveprocedures,whichareassociatedwitha
significantcomplicationrate. Moreover,IAsthathavebeencoiledareattheriskofrecanalization[2,3].
Int.J.Mol.Sci.2018,19,1245;doi:10.3390/ijms19041245 www.mdpi.com/journal/ijms
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Ontheotherhand,a“watchandsee”strategyisoftenemotionallyunacceptabletothosepatientswith
aconstantfearofimpendingbleeding. Hence,studiesonthemolecularmechanismofIAformation,
development,andrupturearejustified. InflammationisusuallydetectedinIAwallsanditcanalso
contributetotheremodelingofthecerebralvesselwallassociatedwithIArupture.Manyproteinscontrol
inflammation,includingthetranscriptionfactorNF-κB(nuclearfactorkappa-light-chain-enhancerof
activatedBcells),whichisimportantnotonlyforinflammatoryprocesses,butalsoforseveralvital
functionsofthecell,includingproliferation.
NF-κB interacts with numerous proteins, including those important for autophagy, a process
of recycling of no longer needed or damaged cellular components. Autophagy is implicated in
the pathogenesis of many serious diseases, including cerebral disorders such as cerebral ischemia
and resulting brain injury [4], traumatic brain injury [5], intracerebral hemorrhage [6], and others.
Some studies show that IA formation and its rupture can be related to the expression of certain
autophagyproteins[7,8]. Therefore,itisjustifiedtoexplorethelinkbetweenIA/SAH,inflammation,
andautophagy,asanelementofIA/SAHpathogenesisandtoassessitspotentialintheprevention,
diagnosis,andtherapyofIA/SAH.
2. IntracranialAneurysm
SaccularIA(furthercalledIA)isastable,localizeddilation(outpouching)ofthearterialwall
resultedfromtheweakeningofitsmuscularlayer[9,10]. SuchIAsarefairlycommonandoccurin
a considerable proportion (up to 10%) in some populations [11]. Intact (unruptured) IA is mostly
asymptomaticandoftendiscoveredbychance. However,itcarriesapotentialriskofruptureandfatal
SAH.AlthoughtheseverityofSAHvaries,itsoverallmortalityrateexceeds50%[11].
ThedevelopmentandruptureofIAdependontheremodelingofvesselwalls,which,inturn,
dependsonthebalancebetweenmatrixdegenerationandregeneration. Theformerisexecutedby
proteases,releasedbymacrophagesandotherinflammatorycells,thelatterisprovidedbysmooth
musclecells. Inflammatorycellscangainaccesstothewallsofaffectedvesselsthroughachannel
formedbytheneovascularizationofthevasavasorum[12]. Therefore,angiogenesis,providingnew
vasa vasorum in cerebral artery wall, can be an early step during IA formation [3,12]. These new
bloodvesselscanbeinfiltratedbyinflammatorycells,mainlymacrophagesandneutrophils,which
can remodel the wall to form a clinically detectable IA [13]. Further action of these cells secreting
metalloproteinasesandotherremodelingenzymes,alongwithyetnon-identifiedfactors,canleadto
IArupture,resultinginSAH[13–18](Figure1). Acomparisonofthewallsoriginatingfromruptured
andunrupturedIArevealedagreaterinvasionofinflammatorycellsintheformerthaninlatter[19].
Although our knowledge of IA pathogenesis is far from completeness, it is established that
inflammationplaysanimportantroleinit[1,20,21].TheformationofIAisinitiatedbyahemodynamically
triggeredendothelialcelldysfunctionfollowedbyaninflammatoryreactionwithelevatedactivityof
theinflammatorytranscriptionfactorNF-κB[22](Figure2). NF-κBdenotesafamilyoftranscription
factors playing an important role in many cellular activities, including proliferation, response to
stress,immuneresponse,apoptosis,andinflammation. Inhumans,thereareatleastfivemembersof
theNF-κBfamily:NF-κB1(p50),NF-κB2(p52),RelA(RELAproto-oncogene,NF-κBsubunit,p65),RelB
(RELBproto-oncogene,NF-κBsubunit),andc-Rel(RELproto-oncogene,NF-κBsubunit)[23].Innormal
condition,NF-κBdimerizesandissequesteredinthecytoplasmbyitsinhibitorIκB(nuclearfactorof
kappalightpolypeptidegeneenhancerinBcellsinhibitor),whichpreventsthenucleartranslocationof
thedimer[24].Instressconditions,NF-κBtranslocatestothenucleus,whereitstimulatesorrepressesthe
transcriptionofmanygenes.
TheactivationofNF-κBinbasal(normal)conditionscanbepromotedbytwoseparatesignaling
pathways, i.e., the canonical and non-canonical pathways, which can function in basal and stress
conditions[25–27]. ThebasalcanonicalpathwayismediatedbytheinhibitoroftheIκBkinase(IKK),
whichinactivatesNF-κB[28,29]. UponphosphorylationanddegradationofIκBwiththeinvolvement
ofNF-κB-inducingkinase(NIK),theNF-κBheterodimerintheformofRelA/p50isactivatedand
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translocatesto thenucleus tocontrolthe transcriptionof assortedgenes. The basalnon-canonical
pathwayInti.s J.m Moel.d Sicai. t2e01d8, b19y, xN IKandIKK,whichproteolyticallyprocesstheproteinp100top3 roof d19u cep52,
whichformsaheterodimerwithRelB,regulatinggenetranscription(Figure3). Inbasalconditions,
pathway is mediated by NIK and IKK, which proteolytically process the protein p100 to produce p52,
thesignalsentbyNIKisweak,asNIKispermanentlybeingdegradedbyE3ubiquitinligases[30].
which forms a heterodimer with RelB, regulating gene transcription (Figure 3). In basal conditions,
Thesebasalpathwayscanbestimulatedbyvariousfactors,butingeneralthecanonicalpathwayis
the signal sent by NIK is weak, as NIK is permanently being degraded by E3 ubiquitin ligases [30].
activateTdhbesye tbuamsalo pranthewcraoyss icsafna bceto srtimαu(lTatNedF αby) vaanrdiouins tfeacrtloerusk, binut 1inβ g(eInLe-r1aβl )th,ea scawnoenlilcaals poatthhwerayc yist okines,
withtheaicntivvoatlevde mbye tnutmoofr tnheecirrosspise fcaicfitocr rαe c(eTpNtFoαr)s a[n3d1 ]i.nTtehrleeunkoinn -1cβa n(IoLn-1iβc)a,l aps awthelwl aasy oitshearc ctiyvtoakteindesw, iththe
contribuwtiiothn thoef ianvliomlvietmedenste otfo thfeliirg sapnedcisfi,ci nreccleupdtoinrsg [3t1h]o. Tsheeb neolno-ncganinongictaol tphaethTwNayF isf aamctiivlyateadn wdiTthN thFe- related
contribution of a limited set of ligands, including those belonging to the TNF family and TNF-related
factors[32,33].
factors [32,33].

Figure 1. Molecular mechanism of intracranial aneurysm (IA) growth and rupture.
Figure 1. Molecular mechanism of intracranial aneurysm (IA) growth and rupture.
Neovascularization (dashed thin red lines) of the vasa vasorum (solid thin red lines) in vascular
Neovascsumloaortihz amtiuosncle( dcealslsh (eVdSMthCisn) erendablleins emsa)corofpthhaegevs aasnad voathseorr iunmflam(smoalitdoryth cienllsr etod pleinneetsra)tein thvea scular
smoothvmesuseslc wlealcl ethllrsou(VghS MtheC lasy)eern oaf benledsotmhealicarl ocpelhlsa (gEeCss)a anndd soetchreetre siunbflsatamncmesa, tmoorsytlyce plrlostetoasepse, ntheutsr atethe
vesselwtahlilnnthinrgo uangdh wtheaekleanyinegr tohfee wnadllo. tShueclhi asltrcuecltlusre( EoCf tsh)ea vnedssesle ccarne treupstuubres,t raenscueltsin,gm ino sstulbyaprarcohtneoaisde s,thus
hemorrhage (SAH), but a trigger, mostly unidentified, is needed to initiate this effect.
thinningandweakeningthewall.Suchstructureofthevesselcanrupture,resultinginsubarachnoid

hemorrhage(SAH),butatrigger,mostlyunidentified,isneededtoinitiatethiseffect.
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Int. J. Mol. Sci. 2018, 19, x 4 of 19

Figure 2. Mechanism of IA formation, progression, and rupture. Hemodynamic stress in a blood
Figure 2. Mechanism of IA formation, progression, and rupture. Hemodynamic stress in a
vessel stimulates inflammation in the endothelial cells (ECs), which promotes lamina degradation
blood vessel stimulates inflammation in the endothelial cells (ECs), which promotes lamina
and evokes changes in vascular smooth muscle cells (VSMCs), including inflammation,
degradationandevokeschangesinvascularsmoothmusclecells(VSMCs),includinginflammation,
neovascularization and matrix remodeling, which together modify the properties of the blood vessel
neovascularizationandmatrixremodeling,whichtogethermodifythepropertiesofthebloodvessel
wall. Permanent inflammation leads to the degeneration of VSMCs, resulting in thinning of the VSMC
wall.PermanentinflammationleadstothedegenerationofVSMCs,resultinginthinningoftheVSMC
layers, increase in their fragility, and finally in IA rupture. Red dashed lines denote vasa vasorum,
layers,increaseintheirfragility,andfinallyinIArupture. Reddashedlinesdenotevasavasorum,
black dashed lines: destroyed lamina, red ovals represent blood cells.
blackdashedlines:destroyedlamina,redovalsrepresentbloodcells.
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Int. J. Mol. Sci. 2018, 19, x 5 of 19

Figure 3. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) can be activated in a
Figure3.NF-κB(nuclearfactorkappa-light-chain-enhancerofactivatedBcells)canbeactivatedina
canonical (left) or non-canonical (right) pathway to stimulate or repress the expression of genes
canonicainlv(loelvfte)do irnn tohen -rceagnuloantiiocna lo(f rcieglhlutl)apr aretshpwonasye ttoo ssttrimessu, liamtemournree rpesrpesosnsthe,e aenxdp inreflsasmiomnaotifogn.e Eniethseirn volved
intherepgauthlawtaioy niso ifnictiealtleudl abry rae slipgoannds ebotounsdtr beys si,tsi mremceputnoer. rSeosmpeo nausteo,pahnadgyi npflroateminms acatnio bne. rEeiltahteedr tpoa thway
is initiabteodth bpaythawlaiygsa, nasd dibscouusnsedd ibny Seicttsiorne 4c.e Tphtiosr f.iguSroem is ea saiumtpolpifihedag reyprpersoentetaitniosnc oannly baendr edloaetse ndott o both
contain many important details. IKB (nuclear factor of kappa light polypeptide gene enhancer in B
pathways, as discussed in Section 4. This figure is a simplified representation only and does not
cells inhibitor); IKK: IκB kinase; mTOR: mammalian target of rapamycin; NIK: NF-κB-inducing kinase;
containmanyimportantdetails. IKB(nuclearfactorofkappalightpolypeptidegeneenhancerin
TNFR: tumor necrosis factor receptor; ROS: reactive oxygen species. Subunits of NF-κB are shaded
Bcellsinhibitor); IKK: IκB kinase; mTOR: mammalian target of rapamycin; NIK: NF-κB-inducing
with the same, light fiolet color. Sharp arrows represent stimulation/induction, while blunt arrows:
kinase;iTnNhiFbiRti:ontu/dmowornrnegeucrlaotsioisn,f daacsthoerdr oevcaelp: tthoer ;nRucOleSu:s.r e activeoxygenspecies. SubunitsofNF-κBare
shadedwiththesame,lightfioletcolor.Sharparrowsrepresentstimulation/induction,whileblunt
arrows:Einlehviabtietdio nac/tdivoawtinonre gouf lNatFio-κnB, dleaasdhse dtoo vthael: ethxeprneussciloenu so.f several pro-inflammatory proteins,
including cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE-2), and molecules which facilitate the
recruitment and adhesion of macrophages, which enter the vessel wall through new vasa vasorum
Elevated activation of NF-κB leads to the expression of several pro-inflammatory proteins,
[15,34]. They secrete further pro-inflammatory molecules and proteins, mainly proteases, which can
includinregmcoydcello tohex ystgruecntausree -o2f t(hCeO wXal-l2 [)3,5–p3r7o].s tTahgel ainnfldaimnmEa2tor(yP rGesEp-o2n)s,e ainn dthem voelseseclu wleasll wleahdisc hto faa cilitate
the recrduisitrmupetinotn oafn tdhe aindtehrensailo enlasotifc lmamaicnrao apnhd atog tehse, pwrohteioclhytiecn dteesrtruthcteionv oefs tsheel vwasaculllatrh erxotruacgehllunlaerw vasa
matrix by matrix metalloproteinases (MMPs), with subsequent IA formation via the upregulation of
vasorum[15,34]. Theysecretefurtherpro-inflammatorymoleculesandproteins,mainlyproteases,
other proteinases and angiogenic factors [38]. The inflammatory response also stimulates the
which can remodel the structure of the wall [35–37]. The inflammatory response in the vessel
phenotypic modulation of vascular smooth muscle cells (VSMCs) from a contractile to a pro-
wall leads to a disruption of the internal elastic lamina and to the proteolytic destruction of the
inflammatory/pro-matrix remodeling phenotype, followed by their degeneration, which may be
vascularcreuxcitarla tcoe IlAlu folarmr matiaotnr aixndb pyromgraetsrsiixonm [3e9t]a. Tllhoep prhoetneointyapsiecs m(oMduMlaPtiso)n, owf VitShMsCu biss aesqsuoceinatteIdA wfitohr mation
viatheuthper eexgpurleastsiioonn ooff portoh-ienrflpamromteaitnoaryse, psraon-mdaatrnixg iroemgeondieclifnagc tgoernses[.3 T8h].e Tehxpereinssfiloanm omf MatMorPys,r weshpicohn sealso
stimulataerse tphreodpuhctesn ooft syupcihc gmenoeds,u ilsa stiigonnifiocfanvta isnc buolathr lsemukooocythtesm aunsdc VleSMceCll sw(iVthSinM thCes v)efsrsoeml/anaeucorynstmra ctileto

apro-inflammatory/pro-matrixremodelingphenotype,followedbytheirdegeneration,whichmaybe
crucialtoIAformationandprogression[39]. ThephenotypicmodulationofVSMCisassociatedwith
the expression of pro-inflammatory, pro-matrix remodeling genes. The expression of MMPs, which
are products of such genes, is significant in both leukocytes and VSMC within the vessel/aneurysm
wall [40]. MMPs digest the extracellular matrix in the arterial wall and cause further vessel damage.
Int.J.Mol.Sci.2018,19,1245 6of20
Subsequentchangesinthemedialayerincludeapoptosis/lossofVSMC,changesinVSMCproliferation,
andthinningoftheaneurysmwall[41].Theresultingcelldeathandvesselwalldegenerationultimately
culminateinIArupture.Therefore,theruptureofIAresultsfromachainofmoleculareventsproducing
changesinthebrainvesselwall(Figure2).Thesechangesmaybemodulatedbyoccasionalendogenous
orenvironmentalinfluencesbut,basically,theyareconsequencesofachainoforderedevents. Inother
words,theactofIArupturecanbetriggeredbyanaccidentalaffair,butonlyaftertheoccurrenceof
somemoleculareventsthatarenecessaryforthesusceptibilityofIAtorupture.
Asmentioned,theinflammatorycellsfindtheirwaytoacerebralaneurysmthroughthevasculatureof
vasavasorum.Theprimarysourceofthesecellsisnotpreciselyknown.Itispossiblethatmacrophagesand
otherinflammatorycellsstimulateneoangiogenesisofvasavasorum. Proinflammatoryfactorsreleased
byinflammatorycellsweakenthewallofthevesseldegradingtheextracellularmatrix,whichcan
leadtothebreakageoftheintegrityofthevessellumen[14]. Thecriticalroleofmacropahgesand
otherinflammatorycellsinaneurysmformationanddevelopmentwasconfirmedinmanystudies
performedonanimalmodelsofIAaswellashumanrupturedandunrupturedIAs[13]. Furthermore,
prostaglandinE2–EP2–NF-κBsignalinginmacrophagesisconsideredasatargetinIAtherapy[42].
3. InflammationinIntracranialAneurysm
The potential role of inflammation in the formation and progression of IA was proposed in
severalstudies[43]. Macrophagesarethoughttobethekeyinflammatorycomponentthatinfiltrate
thewallsofIA.Althoughmacrophageinfiltrationwasobservedinbothrupturedandunruptured
IAs, theintensityofthisprocessseemsbeassociatedwiththelossofsmoothmusclecellsandthe
disruptionofthecollagenlayerintheIAwall[19]. Inaddition,itwasshownthattheincidenceofIAs
formationwassignificantlydecreasedinmacrophage-depletedmice[44]. Thesedatasuggestthatthe
formationandruptureofIAsisrelatedtomacrophagesaccumulation.
T lymphocytes are other inflammatory cells detected in IA walls [19]. A positive correlation
wasfoundbetweenthenumberofTcellsandIArupture. Tlymphocytesactivelyparticipateinthe
inflammatory reaction, but it is still unclear whether their increased infiltrations is the cause or a
consequenceoftherupture.
NF-κB has been identified as a regulatory transcription factor of genes induced in IA lesions
and as a possible factor related to IA rupture [40] (Figure 4). Moreover, shear stress—an initiator
of IA formation—was demonstrated to induce the expression of pro-inflammatory genes via an
NF-κB-mediatedpathway[45].NF-κBover-activationleadstotheexpressionofinflammatoryproteins,
including cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE-2), and others, which stimulate IA
progression[46]. NF-κBalsoinducestheexpressionofMCP-1(monocytechemoattractantprotein1)
andVCAM-1(vascularcelladhesionmolecule1),responsiblefortherecruitmentandadhesionof
macrophages,whichpenetratethevesselwallthroughjunctionsbetweenendothelialcellsandnew
vasavasorum[15,34]. Themacrophagesreleaseotherpro-inflammatorymolecules,includingtumor
necrosisfactorα(TNF-α),interleukin1β(IL-1β),metalloproteinases,aswellasotherproteases,which
remodelthestructureofthewallinconcertwithothermolecules[35–37]. NF-κBwasactivatedin
endothelialcellsandmacrophagesinIAwallsduringIAformation[47]. Thedeficiencyorinhibitionof
NF-κBsignificantlyreducedtheexpressionandproductionofdownstreampro-inflammatoryfactors
and suppressed IA formation and progression in rodent models [46,48]. NF-κB is also a mediator
of the downregulation of procollagen type I and III gene expression, which was observed in IA
patients[46]. NomutationwasfoundintypeIIIprocollagengeneinpatientswithIA,thusthereduced
productionoftypeIIIcollagenmaybederivedfromimpairedNF-κB-dependentregulationofthe
expressionofthisgene. SeveralcytokinesareassociatedwithIAformation. Monocytechemoattractant
protein1(MCP-1)isachemokinethatmodulatesthemigrationandinfiltrationofmonocytesandT
lymphocytes. AnincreasedexpressionoftheMCP-1genewasfoundinthewallofIAattheearly
stageofitsformationinrats[34]. Moreover,MCP-1knockoutmiceshowedasignificantdecreasein
macrophageaccumulationandareducedincidenceofIA,suggestingthatMCP-1maybeacritical
Int.J.Mol.Sci.2018,19,1245 7of20
elementinaneurysmpathogenesis[34]. VCAM-1isotherinflammatorycomponentthatparticipates
Int. J. Mol. Sci. 2018, 19, x 7 of 19
intherecruitmentofmonocytes. Italsoregulatestheadhesionofmonocytestoendothelialcellsinthe
vesselwdaecllr.eaAsen inin mcarceraospehdageex apccruemssuiolantioonf aVndC aA rMed-u1ceidn ihncuidmenacne IoAf IAca, ssuegsgwesatisngd tehsact rMibCePd-1[ m49a]y. bCey at okines,
includincrgitiMcalC ePle-m1,eSntD iFn- 1an(eCu-rXys-mC mpaothtoifgecnheesims o[3k4i]n. eVCliAgaMn-d1 i1s2 o)tahnerd inVflCamAmMat-o1rya rceomrepsopnoennts itbhlaet for the
participates in the recruitment of monocytes. It also regulates the adhesion of monocytes to endothelial
recruitmentofmacrophagesintothewall,thustheymayinitiatetheinflammatoryreactionandwall
cells in the vessel wall. An increased expression of VCAM-1 in human IA cases was described [49].
remodeling. Therefore,itseemsthatcytokinesplayakeyroleinaneurysmformationandprogression,
Cytokines, including MCP-1, SDF-1 (C-X-C motif chemokine ligand 12) and VCAM-1 are responsible
andtheiractioncanleadtotheactivationofNF-κB(Section2andFigure2).
for the recruitment of macrophages into the wall, thus they may initiate the inflammatory reaction and
Altwhaolul grehmtohdeelrionlge. oTfheinreflfoarme, mita steioemnsi nthIaAt dcyetvokeilnoepsm pelanyt ias kneoyt rfoullel yinu nandeeurrsytsomo dfo,rtmheataiobno vanedfi ndings
suggestptrhoagtreNssFio-κn,B ancadn thbeeir carcutiocina clatno laeande utor tyhsem acftoivramtioanti oofn NaFn-κdBp (Sroecgtiroens s2i oannd. FTihgeurien 2fl).a mmatoryresponse
involvingleAukltohocyugtehs thaen rdolcey otfo iknifnlaemsmseaetimons into IAb edeavsesloocpimateendt iws niotht ffuullnyc utniodnerasltoaondd, tmheo arbpohvoe lfoingdicinaglsc hanges
suggest that NF-κB can be crucial to aneurysm formation and progression. The inflammatory
inthevesselwallthatleadtoIA.
response involving leukocytes and cytokines seems to be associated with functional and
Because of a low availability of human IA specimens, the studies on IA pathogenesis has
morphological changes in the vessel wall that lead to IA.
been mainly conducted on animal models of IA, which strongly support a crucial role of the
Because of a low availability of human IA specimens, the studies on IA pathogenesis has been
inflammmaationrlyy croensdpuocntesde oinn atnhiemianl tmraocdrealns oiaf lIAar, twehriiachl wstraolnlsgliyn sIuAppfoorrtm a cartuiocinal arnolde opfr tohge riensflsaimonm.aAtomry ongthe
factorsrreesgpuolnastei ning itnhefl ainmtrmacraatnoirayl aprrteorciaels sweasl,lsN iFn -κIAB fcoarnmbateioonn aenodf ptrhoegrmesosisotns. igAnmiofincga nthtep rfaoctteoirns sinthe
pathogernegeusilastionfgI Ainffloarmmmaattioorny apnrodcersuspest,u NreF[-4κ1B] .can be one of the most significant proteins in the
pathogenesis of IA formation and rupture [41].

Figure4F.igMuroel e4c. uMlaorlemcuelcarh amneicshmanoisfmin otrf aicnrtaranciraalnaianl eaunreyusrmysm(I A(IA)f)o fromrmaatitoionn aanndd pprrooggrreessssioionn. .AA wwalal llshear
shear stress can be an initial trigger in IA formation. This stress can result in some fragile sites (Figure
stresscanbeaninitialtriggerinIAformation. Thisstresscanresultinsomefragilesites(Figure1)
1) to which macrophages can be recruited by MCP-1 (monocyte chemoattractant protein 1), which is
towhichmacrophagescanberecruitedbyMCP-1(monocytechemoattractantprotein1),whichis
expressed after NF-κB activation, resulting also in the expression of other pro-inflammatory proteins,
expressedafterNF-κBactivation,resultingalsointheexpressionofotherpro-inflammatoryproteins,
including COX-2 and PGE-2, in endothelial cells (ECs). Macrophages, which adhere to ECs with the
includiningvColvOeXm-e2nta onfd VPCGAME--21 ,pirnoteenind, fointhde tlhieailr cwealyls to(E thCes v)e.sMsela wcraollp thhraoguegsh, nwewhi vcahsaa dvahseorreumto, sEecCrestew iththe
involvemotheenrt porfoV-inCflAamMm-1atporryo tperionte,ifinns,d intchlueidrinwg aTyNtFo-αth, eILv-1eβs,s aenldw malaltrtihxr oreumgohdenleinwg vpraostaeivnsa ssourcuh mas, secrete
otherprmoa-tirnixfl mametamllaoptorortyeipnarsoetse MinMs,Pi-n2 calnudd MinMgPT-9N. NFF-α-κ,BI Lac-t1ivβa,tiaonn dis mmeadtiraitxedr ebmy IoKdKe (lIiκnBg kpinraostee) iannsd suchas
NIK (NF-κB-inducing kinase) proteins, whose activity is regulated by autophagy. The concerted
matrixmetalloproteinasesMMP-2andMMP-9.NF-κBactivationismediatedbyIKK(IκBkinase)and
action of these proteins can result in a further weakening of the wall structure and, eventually, in its
NIK(NF-κB-inducingkinase)proteins,whoseactivityisregulatedbyautophagy.Theconcertedaction
rupture, but the action of some, yet unidentified factors, can be the final trigger of IA rupture. Black
oftheseproteinscanresultinafurtherweakeningofthewallstructureand,eventually,initsrupture,
and red solid/dashed lines denote normal/disrupted lamina and vasa vasorum, respectively.
buttheactionofsome,yetunidentifiedfactors,canbethefinaltriggerofIArupture.Blackandred
solid/dashedlinesdenotenormal/disruptedlaminaandvasavasorum,respectively.
Int.J.Mol.Sci.2018,19,1245 8of20
4. NF-κBSignalingPathwaysandAutophagy
Besidesactingasatranscriptionfactor,NF-κBcanmodulateseveralsignalingpathways.Thelevel
ofautophagyiscontrolledpositivelyornegativelybyNF-κBthroughitsinducers. NF-κBactivation
byTNFinhibitsautophagybytheactivationofthemTOR(mechanistictargetofrapamycinkinase)
complexandtheinhibitionofreactiveoxygenspecies(ROS)[50]. TRAF6(TNFreceptor-associated
factor 6), a member of the TRAF family, can act as a signal transducer in the NF-κB signaling
pathway[51]. ItcanalsoinduceubiquitinationofBeclin1,akeyproteinofautophagy,ifstimulated
bytoll-likereceptor4(TLR4)[52]. However,Beclin1ubiquitinationisrepressedbyA20,anNF-κB
inhibitor,resultinginautophagyinhibition[53]. Autophagycanbeinducedinhypoxicconditionsby
hypoxia-inducedfactor1α(HIF-1α),butthisinductionisrelatedtotheactivationofstress-responsive
transcriptionfactors,includingNF-κB[54]. ItwasshownthatautophagyactivationbyNF-κBwas
essentialforthecelltosurviveafterheatshock[55]. Moreover,themodulationofautophagybyNF-κB
playedaroleincellsurvivalinresponsetoproteinaggregationstress,andthisreactionseemedtobe
independentoftheprimarystressfactor[56,57].
RelA(p65),amemberoftheNF-κBfamily,cantargetseveralsitesinthepromoteroftheBECN1
(Beclin1)gene,modulatingthelevelofautophagyinvarioushumancelllines[58,59]. Inturn,other
membersoftheNF-κBfamilycanbeactivatedbyDNAdamagewiththeinvolvementofautophagy,
asreportedformanycelllinesandseveralsignalingproteins[60–65].Bcl-2/E1B-19Kinteractingprotein
3(BNIP3)isamitochondrialproteincooperatingwithseveralanti-apoptoticproteinsandinducing
celldeathwiththeinvolvementofautophagy[66,67]. RelA(p65)wasreportedtoblockthebinding
oftheBNIP3promoter, andthiseffectalsocanbelinkedwithamutualcontrolofNF-κB-directed
inflammationandautophagy[68,69]. Transforminggrowthfactor-β-activatingkinase1(TAK1)can
activateautophagybytheinhibitionofmTORC1(mechanistictargetofrapamycincomplex1),inan
analogousfashiontoIKKsinstarvedcells[54,70]. However,itisnotcompletelyclearwhetherNF-κB
isneededforsuchTAK1action[71].
Theinflammasomeisalargesignalingcomplexresponsiblefortheactivationofinflammatory
processes. It can be formed in response to infection, disturbed metabolism, and tissue damage
(Figure 5). The inflammasome contains caspase-1, which is an inflammatory protease involved in
pyroptosis,avariantofprogrammedcellulardeath,andproteolyticcleavageoftwoinflammatory
cytokines, namely, IL-1β and interleukin 18 (IL-18) [72]. The inflammasome can be formed in
responsetothecytosolicrecognitionofDAMPs(damage-associatedmolecularpatterns)andPAMPs
(pathogen-associated patterns) by nod-like receptors (NLRs) [73]. The inflammasome contains
also PYCARD (apoptosis-associated speck-like protein containing a caspase recruitment domain),
which is essential for its activation. Probably the best studied and known inflammasome is the
NLRP3(NLR(nucleotide-bindingoligomerizationdomain,leucinerichrepeat)familypyrindomain
containing3)inflammasome[74,75].TheactivityofNLRP3isinducedbyavarietyoffactors,including
potassiumions,cholesterol,mitochondrialROS,amyloid,fattyacids[76]. TheNLRP3inflammasome
isprimedbyTLRsignalingpathwaywiththeinvolvementofNF-κB[77]. Autophagycanregulatethe
inflammasomebyamechanismdependentonNLR,controllingthelevelofinflammatorycytokines,
IL-1βandIL-18[78–80]. AutophagycandegradeNF-κBsignalingproteinsIKKandNIK,andthis
process is mediated by the inhibition of Hsp90 (heat shock protein 90) protein [81]. In general,
autophagy can specifically degrade NF-κB signaling components, and this effect can result in the
cessationofNF-κBactivationorinitsstimulation,whensignalinginhibitorsaredegraded[71].
Int.J.Mol.Sci.2018,19,1245 9of20
Int. J. Mol. Sci. 2018, 19, x 9 of 19

Figure 5. The inflammasome and the interplay between autophagy and NF-κB-mediated
Figure5.TheinflammasomeandtheinterplaybetweenautophagyandNF-κB-mediatedinflammation.
inflammation. See the text for more details. PYCARD (apoptosis-associated speck-like protein
Seethecteoxnttafionirnmg ao rceasdpeatsae irlsec.rPuYitmCeAnRt dDom(aapino)p; tDoAsiMs-Pa:s dsaomciaagtee-dasssopceiactke-dl imkeolpecruoltaeri npactotenrnta; iInKiKn:g IκaBc aspase
recruitmkeinnatsed; oILm: aininte)r;leDuAkinM; PN:Fd-κaBm: naugcele-aars sfoacctioart ekdapmpao-lliegchut-lcahrapina-ettnehrann;ceIKr oKf :aIcκtiBvakteinda Bs ec;eIllLs;: NinItKe:r leukin;
NF-κB:NnuF-cκlBe-airndfauccitnogr kkinaapspe;a N-lLigRhPt3-:c NhLaRin f-aemnihlya pnycreinr doofmaacitniv caontetadinBincge 3l;l sP;ANMIPK: :pNathFo-gκeBn--iansdsoucciaitnegd kinase;
pattern; TLR4: Toll-like receptor 4.
NLRP3:NLRfamilypyrindomaincontaining3;PAMP:pathogen-associatedpattern;TLR4:Toll-like
receptor4.
It was shown that inhibition of NF-κB could stimulate autophagy via the JNK (c-Jun N-terminal
kinase) signaling pathway in porcine granulosa cells [82]. Activation of the JNK pathway and
Itwoavsersehxopwrensstihona toifn hBiebcilitnio 1n wofeNre Fr-eκpBorctoedu lidn satuimtouplhaatgeya uintdoupcheadg byyv ciaertahmeiJdNe Kin( ch-uJmunanN h-teepramtoimnaal kinase)
Hep3B cells [83].
signalingpathwayinporcinegranulosacells[82]. ActivationoftheJNKpathwayandoverexpression
ofBeclin1werereportedinautophagyinducedbyceramideinhumanhepatomaHep3Bcells[83].
5. Autophagy and Its Potential Involvement in Intracranial Aneurysm and Aneurysmal SAH
5. AutophagAyutaonpdhaIgtys P(soeltfe-enattiianlg)I ninv oitlsv ebmasiecn ftoirnm Iins tara pcrroacneisas loAf nlyesuosroymsmal adnegdraAdnateiounr yosf mceallluSlaAr H
components which are no longer needed or are damaged. This degradation can yield elements which
Auctaonp hbea guysed(s eblyf -tehaet icnegll) tion pirtosdubcaes iecnefrogrym anids/oar pcerlolucleasrs boufildlyinsgo sbolomckasl. Adeugtorpahdaagtyi ocnano bfec ellular
componsetinmtsulwathedic bhya drieffneroenlto sntgimerulni,e iendcleuddionrg astraervdaatimona.g Tehde.reTfohries, dauetgorpahdaagtyi ocannc baen cyoniesliddeereledm ase nants which
canbeu sedbythecelltoproduceenergyand/orcellularbuildingblocks.Autophagycanbestimulated
bydifferentstimuli,includingstarvation. Therefore,autophagycanbeconsideredasanimportant
elementincellularmetabolism,aswellasapartofcytoplasmicqualitycontrol. Thesebasicfunctions
ofautophagycanbeextendedbyitsactionagainstexternalinvaders,theregulationofprogrammed
cellulardeath,itsinvolvementinDNAdamageresponse,andothers[84]. Disturbedautophagycanbe
Int.J.Mol.Sci.2018,19,1245 10of20
associatedwithhumandisorders,includingcancer,immunologicalandneurodegenerativediseases,
eyedefects,andmanyothers[85].
Autophagycanbedividedintothreeclasses.Macroautophagyhelpsthecelltosurviveinunfavorable
energetic and/or nutritional conditions. The material to be degraded (cargo) is packaged into a
membranedstructure,theautophagosome,whichisthentransportedintolysosomesfordegradation.
Inmicroautophagy,thecargoisdirectlyengulfedbythelysosomes.Inchaperone-mediatedautophagy,
proteinswithaspecificaminoacidmotif,withtheinvolvementofHSC70(heatshockproteinfamilyA
(Hsp70)member8)protein,achaperone,andseveralofitspartners,aredegraded.
Macroautophagy,furtherreferredtoasautophagy,requiresajointactionofautophagosomeand
lysosome to degrade and recycle the cargo. Lysosomal degradation occurs with the involvement
of many autophagy-related proteins (ATGs). Autophagy can be categorized on the basis of
pivotalsignalingpathwaysinvolvedinitsregulation—ingeneral,asmTOR(mammaliantargetof
rapamycin)-dependentand-independent[86]. Intheformer,theinactivationofmTORisthedecisive
eventopeningthepathwaytoformanisolationmembranethatwillencirclethecargo,thusformingthe
autophagosome,whichfuseswiththelysosometoproducetheautolysosome,inwhichtherecycling
ofthecargooccurs(Figure6). Thischainofeventsissupportedbyanumberofproteinsundergoing
activationandmodifications. TheseincludeATG13,ATG101serine/threoninekinases(ULKs),LC3
(microtubule associated protein 1 light chain 3α), p62 (SQSTM1, sequestosome 1), and the main
phospholipidusedtobuildtheautophagosome,phosphatidylethanoamine(PE).mTOR-independent
autophagy in mammals was firstly described by Sarkar et al., who showed that lithium induced
autophagymediatedbytheinhibitionofinositolmonophosphataseandresultingintheloweringof
intracellularinositolorinositol1,4,5-trisphosphate(IP3)levels[87]. Manyothersmallmoleculeswere
reportedtoinduceautophagyinanmTOR-independentfashion[86,88].
Asmentioned,autophagycanbeinvolvedinthepathogenesisofmanyhumandiseases.However,
itsinvolvementinIAandSAHisamatterofdebate.ToconsiderthepotentialofautophagyinIA/SAH,
itisreasonabletoexploretheroleofautophagyinnormalvessels,asbasalautophagyisanimportant
mediatorofnormalvascularfunctionanditcanplayaprotectiveroleagainstdamagetothevessel
wallcells,includingendothelialandsmoothmusclecells[89,90]. However,suchroleofautophagy
can be performed by proapoptotic mechanisms. Autophagy can be involved in the proliferation
andacquisitionofasyntheticphenotypebysmoothmusclecells[91–93]. Researchonexperimental
animalsshowedthatdefectiveautophagyinsmoothmusclecellscouldsupportprematuresenescence
inresponsetoastressfactor[94]. Therefore,itistemptingtospeculatethatimpairedautophagycan
contributetoasenescentphenotypeinthearterialwalls,correspondingtoagedvessels,inwhichthe
ratioofpathologicaleventsishigher(Figure7). Thiscouldbeseeninawidercontext,exemplified
byworkshowingthatimpairedautophagycanalterthemorphologyofvesselsmoothmusclecells
andimpacttheirstructuralandfunctionalproperties. Rapamycinanditsanalogs,whicharepotent
inducers of autophagy, are undergoing a number of clinical trials in vascular diseases, and stents
coatedwiththesedrugshaverevolutionizedinterventionalcardiology[95].
Asbasalautophagyisamechanismtosurviveforvesselsmoothmusclecells,itsstress-induced
disturbance can result in the death of these cells, which, in turn, can lead to the weakening of the
mechanisticpropertiesofthewallandtoitslocalrupture. Thiswouldexplainthecontributionof
impairedautophagytoSAHresultingfromIArupture. However,impairedautophagycanbereplaced
by other protective mechanism(s), and this replacement could result in an increased protection,
as shown by Grootaert et al. [94]. In that work, mice with a double deletion of the ATG7 gene,
essentialforbasalautophagy,weremoreresistanttooxidativestressthantheirwild-typecounterparts.
ThiseffectwasunderlinedbythestimulationofphaseIIenzymesGSTA(glutathioneS-transferase)
andNQO1(NAD(P)Hquinonedehydrogenase1)bytheSQSTM1protein,overexpressedincellswith
disturbedautophagy[96].