Nox, Reactive Oxygen Species and Regulation of Vascular Cell Fate

22 Pages·2017·2.42 MB·English

antioxidants
Review
Nox, Reactive Oxygen Species and Regulation of
Vascular Cell Fate
DeniseBurtenshaw1 ID,RoyaHakimjavadi1,EileenM.Redmond2 ID andPaulA.Cahill1,*
1 VascularBiology&Therapeutics,SchoolofBiotechnology,DublinCityUniversity,D09NR58Dublin,Ireland;
[email protected](D.B.);[email protected](R.H.)
2 DepartmentofSurgery(Research),UniversityofRochesterMedicalCentre,Rochester,NY14620,USA;
[email protected]
* Correspondence:[email protected];Tel.:[phone]
Received:2October2017;Accepted:7November2017;Published:14November2017
Abstract: The generation of reactive oxygen species (ROS) and an imbalance of antioxidant
defence mechanisms can result in oxidative stress. Several pro-atherogenic stimuli that promote
intimal-medialthickening(IMT)andearlyarterioscleroticdiseaseprogressionshareoxidativestress
asacommonregulatorypathwaydictatingvascularcellfate. ThemajorsourceofROSgenerated
withinthevascularsystemisthenicotinamideadeninedinucleotidephosphate(NADPH)oxidase
familyofenzymes(Nox),ofwhichsevenmembershavebeencharacterized. TheNoxfamilyare
criticaldeterminantsoftheredoxstatewithinthevesselwallthatdictate,inpartthepathophysiology
ofseveralvascularphenotypes.ThisreviewhighlightstheputativeroleofROSincontrollingvascular
fatebypromotingendothelialdysfunction,alteringvascularsmoothmusclephenotypeanddictating
residentvascularstemcellfate,allofwhichcontributetointimalmedialthickeningandvascular
diseaseprogression.
Keywords:Nox;ROS;vascularsmoothmuscle;endothelial;stemcells;adventitialcells;arteriosclerotic
disease;intimal-medialthickening
1. Introduction
The major reactive oxygen species (ROS)-producing systems within the vascular bed include
thereducedformofnicotinamideadeninedinucleotidephosphate(NADPH)oxidase[1],xanthine
oxidase[2],themitochondrialelectrontransportchain[3],anduncoupledendothelialnitricoxide
(NO)synthase[4]. TheNoxfamilyofNADPHoxidasesareanimportantsourceofROSandarecritical
determinantsoftheredoxstateofthevesselwall. ChangesintheexpressionofNoxcandictate,inpart,
thepathophysiologyofseveralvascularphenotypessinceROSproductionmayexceedtheavailable
antioxidantdefencesystems,therebyfacilitatingoxidativestress[5].
ROScanbeeitherdysfunctionalorprotective. Superoxideanions(O −)reactwithNOtoform
2
peroxynitrite(ONOO−)thatcausesnitricoxidesynthase(NOS)uncouplinginendothelialcells(EC)
andlimitstheamountofNOthatisprotective. Peroxynitrite(ONOO−)alsoinducesthioloxidationas
wellastyrosinenitrationcausingfurtherdamagetothevasculature[6]. Moreover,thedismutation
productofO −,hydrogenperoxide(H O ),directlypromotesvascularsmoothmusclecell(vSMC)
2 2 2
hypertrophy, activates metalloproteinases, and at higher concentrations, inhibits endothelial NOS
(eNOS) by phosphorylation of tyrosine 657 through the redox-activated tyrosine kinase Pyk2 [7].
H O canalsoactivateproteinkinase-GIαtoinducethioloxidationandsubsequentdimerization[8].
2 2
Insomeinstances,H O maybeprotectiveandactivateeNOStocounteractthepro-atherogeniceffects
2 2
ofvariouspathologicalstimuli[8].
Arteriosclerosisisanimportantage-dependentconditionthatincludesatherosclerosis,pulmonary
hypertension,in-stentrestenosis,autologousbypassgraftingandtransplantarteriosclerosisandmay
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resultinaheartattackorstroke[9]. Ahallmarkofarteriosclerosisisapathologicvascularfibrosis
duetotheaccumulationofvSMC-likeneointimalcellsresultinginintimal-medialthickening(IMT)
and lesion formation that significantly narrows the vessel lumen but also provides a substrate for
lipoproteinretentionleadingtoacceleratedatherosclerosis[9].
Animal studies have provided compelling evidence supporting a role for oxidative stress in
contributingtoIMTandtheprogressionofearlyarterioscleroticdisease[6]. Sincevascularlesions
developpreferentiallywithinregionsexposedtodisturbedbloodflowpatternsresultinginenhanced
oxidativestress,theroleofROSincontrollingendothelialdysfunctionduringtheinitiationofvascular
pathologies has attracted extensive interest [10]. Subsequently, the important modulatory role of
ROS in controlling de-differentiation and reprogramming of vSMCs [11] in addition to myogenic
differentiationofresidentvascularstemcellniche(s)andtransforminggrowthfactorβ1(TGF-β1)
inducedendothelial-mesenchymalstemcelltransition(EndoMT)hasemerged[12–14].
Stem cells play an important role during developmental stages due to their unique ability to
self-renewanddifferentiate. However,theirputativeroleinvasculardiseaseprogressionhasonly
recently attracted considerable interest [15]. Many types of stem cells can be cultivated invitro
including embryonic (ESCs), pluripotent (iPSCs), and mesenchymal (MSCs), each with the ability
to differentiate into vascular cell lineages via progenitor cells [16]. In the past decade, numerous
studieshavedemonstratedmyogenicdifferentiationofmesenchymal-likestemcellstovSMClineages
invitro following appropriate inductive stimulation [17], and invivo, using transgenic Cre-LoxP
markedcellsafteriatrogenicinjuryinmurinemodelsofIMT[12,18,19]. However,littleisknownabout
themetabolicchangesoccurringduringthemyogenicdifferentiationprocess[20]. Severalstudies
have demonstrated the importance of the regulation of redox states by Nox isoforms in stem cell
maintenance,proliferationanddifferentiation[21].
Cardiovascularriskfactorssuchashypercholesterolemia,hypertension,diabetesmellitusand
smokingallenhanceROSgenerationandexacerbatethedecreaseinendothelialNOproduction[6].
Moreover,keymoleculareventsduringvasculardiseaseprogressionsuchasoxidativemodificationof
lipoproteinsandphospholipids[22],ECactivationandpermeabilitychanges[23]anddisruptionof
theglycocalyx[24]andcellularinfiltration/activationarefacilitatedbyvascularoxidativestress[5].
It is commonly recognised that high levels of ROS may have destructive effects on both
differentiatedvSMCandEC[25,26]. ThisreviewwilldiscusstheimportanceofNoxgenerationof
ROSincontrollingEC,vSMCandmesenchymal-likestemcellfunctionandfateandtheircontribution
toIMTandvasculardiseaseprogression.
2. VascularNox/DuoxIsoforms
Low to moderate levels of ROS are known to contribute to important functions such as cell
differentiation, migration, adhesion, senescence, growth, and apoptosis. In contrast, a variety of
cardiovasculardiseasesareassociatedwithelevatedROSlevels. AsignificantsourceofcellularROSis
theNoxfamilyofenzymes[27].Initially,theoverproductionofROSsuchasO −,H O ,NO,hydroxyl
2 2 2
radicals, ONOO− and other metabolites within a cell system was labelled as a random event [28].
However, in recent years, studies have confirmed that ROS is a controlled signalling system with
enzymesNoxanddualoxidase(Duox)attheforefront[29,30].
In general, mammals have seven isoforms of Nox enzymes; Nox1, 2, 3, 4 and 5, Duox1 and
Duox2. ThesevenclassifiedNoxproteinsarepresentnotonlyintheplasmamembrane,(Nox1to5
andDuox1/2),butalsointheendoplasmicreticulum,(Nox2,4and5),themitochondrialmembrane
(Nox4)andthenuclearmembrane,(Nox4and5)[31]. Withinthevasculature,Noxisoformsarefound
inECs[32],vSMCs[33],adventitialfibroblasts[34]andstemcells[35](Figure1).
All Nox enzymes are heteroprotein transmembrane complexes (except Nox5) with specific
regulatorymechanisms,tissuespecificityanddownstreamtargetsandfunctions. Theycontainsix
transmembrane domains with conserved binding sites for flavin adenine dinucleotide (FAD) and
NADPHandfourhaem-bindinghistidinesinthethirdandfifthtransmembranedomainstoproduce
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ROSbytransferringanelectrontomolecularoxygen[36]. EachmemberoftheNoxfamilyretains
thecatalyticdomainallowingthetransferofelectronsfromcytosolicNADPHduringthegeneration
ofROSmetabolites. ThistransferleadstothereductionofoxygenbyNox/DuoxtoO − aswellas
2
tosecondaryROSby-products[37]. Duringthisprocess,twoO − moleculeshavethepotentialto
2
reactgeneratingH O ,eitherspontaneously,ordrivenbytheenzymesuperoxidedismutase(SOD)
2 2
inaprocesscalleddismutation. H O canfurtherreactwithO − inthepresenceofiron,generating
2 2 2
ahydroxylradical. Inaddition,otherROSproductscanbegeneratedincludingtheproductionof
hypochlorous acid from the reaction of H O and chloride driven by the presence of the enzyme
2 2
myeloperoxidase or the production of ONOO− generated by the reaction of O − with NO [38].
2
Theresultingbi-productsofthesechainreactions(H O ,NO)possessbeneficialfunctionsinmicrobial
2 2
killing,cellsignalling,differentiationandgeneregulation. However,otherROSproductsincluding
hydroxylradicals,hypochlorousacidandONOO− aremoreoftenassociatedwithcellulardamage
andmolecularpathologiesinextremeconditionsofoxidativestress[38].
TheprimaryROSproductofNox1,2,3and5isO −;however,Nox4,Duox1andDuox2produce
2
mainlyH O [39].Despitesimilaritiesincorestructures,Noxhomologueshavedifferentmechanismsof
2 2
activation.Nox2isthemostclearlydefined[40].Onactivation,p47phoxisphosphorylatedandtranslocates
to the membrane by forming a complex with p67phox and p40phox. Phosphorylation of p47phox
inducesaconformationalchangeinatandemSrchomology3(SH3)domainthatfacilitatesbinding
toaproline-richregioninthecytosolicC-terminusofthetransmembranesubunitp22phox[41,42].
Independently,theguanosine-5(cid:48)-triphosphateGTP-bindingproteinRas-relatedC3botulinumtoxin
substrate (Rac) also moves to the membrane to enable activation. Nox1 also requires association
withcytosoliccomponents(p47phox,p67phoxorNoxO1andNoxA1). Incontrast,theNox4-based
oxidaseappearstobeconstitutivelyactiveanddoesnotrequirep47phox,p67phox,orRac. Recent
studies suggest that Poldip2, a polymerase (DNA-directed) delta interacting protein-2 is a novel
Nox4/p22phoxinteractingproteinthatpositivelyregulatesNox4[43]. TheNox4/p22phox/Poldip2
complexhasprofoundeffectsonRho-dependentcytoskeletalreorganizationandcellularmigration
andmaybefundamentaltothephysiologicalandpathophysiologicalroleofNox4[43]. Duox,Duox2
andNox5requireacalciumionbindingthroughthepresenceofahelix-loop-helixstructuraldomain
(EF-hand) [31] and are activated by an elevation in intracellular Ca2+ [44,45]. Once bound to the
required membrane proteins, the activated Nox/Duox enzyme can successfully transfer electrons
acrossthemembrane[46].
In addition to being highly reactive and oxidizing proteins, DNA and lipids, ROS are now
considered important signalling molecules in their own right, modulating cellular processes such
asgeneexpression,proliferation,andmigration. Importantly,Noxenzymesaretheonlyenzymes
whoseprimaryfunctionistogenerateROS[36]. Otherenzymes(cyclooxygenases,cytochromeP450,
enzymesofthemitochondrialelectrontransportchain)canproduceROS,butonlyasaby-product
of their normal function. In addition, secondary sources of ROS are generated from “uncoupled”
eNOSandxanthineoxidase,whicharedysfunctionalvariantsofeNOSandxanthinedehydrogenase,
respectively[36].
NumerousstimulipromotesustainedactivationofvascularNoxincludingseveralgrowthfactors
(platelet-derivedgrowthfactor(PDGF)[47],epidermalgrowthfactor(EGF)[48],andTGF-β1[49]),
cytokines [50], mechanical forces (cyclic stretch, laminar, and oscillatory shear stress) [51],
metabolic factors (hyperglycemia, hyperinsulinemia, free fatty acids, and advanced glycation end
products)[52],andGprotein–coupledreceptoragonists(serotonin,thrombin,bradykinin,endothelin,
and angiotensin II [Ang II]) [53]. Signalling proteins, c-Src, p21Ras, protein kinase C (PKC),
phospholipase D and phospholipase A (PLA ) all play key roles in vascular NADPH oxidase
2 2
activation[54]. MultiplepathwaysofNoxactivationhavebeendescribedinvariouscelltypesunder
normalandpathologicalstatesincludingphosphorylationofcytosolicregulatorysubunitsbyprotein
kinase C (PKC), protein kinase A (PKA), phosphatidylinositol-3-kinase (PI3K), mitogen-activated
proteinkinases(MAPK),andnon-receptorassociatedproteinkinases(e.g.,Januskinase(JAK)andthe
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non-receptortyrosinekinases,Src)[55].Transientchangesinintracellularionfluxesandprotein-protein
interactionswithmembersofthethioredoxinfamilymayalsopromoteNoxactivity[55].
RedoxregulationofvascularNoxactivationprovidesimportantpositiveandnegativefeedback
regulatory mechanisms [56]. Cellular redox status is maintained by intracellular redox-regulating
molecules, including thioredoxin (TRX). Nuclear factor erythroid 2–related factor 2 (Nrf2),
anantioxidanttranscriptionalfactorisregulatedbytheintracellularredoxstate. BindingsitesforNrf2
arelocatedinthepromoterregionofalargevarietyofgenes,includingNox4[55]. Theseself-limiting
negativefeedbackmechanismspredominateduringphysiologicconditionsbutmayalsobeinvolved
inmaintaininglowoutputofthenonphagocyteNox,whereasapositivemechanismmaypredominate
inavarietyofvasculardiseases[36].
Figure1. Noxenzymeswithinthevasculature. TheschematicdepictstheNoxenzymeswithinthe
vascularwall.Allthreelayersofthevascularwall[intima(i.e.,endothelialcells),media(i.e.,smooth
musclecells),andadventitia(i.e.,fibroblastsandmacrophages)]expressNoxfamilymembers.Nox4
is the predominant isoform in endothelial cells, Nox1 and Nox4 in smooth muscle cells, Nox4 in
fibroblasts,andNox2andNox4instemcells.Nox-derivedO −avidlyreactswithNO.Nox-derived
2
ROS also affect the extracellular matrix and the external (EEL) and internal elastic lamina (IEL),
influencegeneexpressionandareinvolvedincellproliferation,migrationanddifferentiationthatlead
tovasculardiseaseprogression[55].
3. NoxandVascularEndothelialCells
TheroleofNoxisoformswithintheintimalendotheliallayerhasbeenthesubjectofseveralstudies.
Thevascularendotheliumplaysacriticalroleincardiovascularhomeostasis. Vascularendothelialcells
normallyperformkeyhomeostaticfunctionssuchasmaintainingbloodfluid,regulatingbloodflow,
regulatingmacromoleculeandfluidexchangewiththetissuesandpreventingleukocyteactivation[57].
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Endothelial dysfunction is characterized by a shift in the actions of the endothelial cell towards
areducedendothelium-dependentvasodilation(viareducedbioavailabilityofNOandprostacyclin)
andpromotionofinflammatoryandprothromboticstate[57]. Thesechangesareassociatedwithmost
formsofcardiovasculardisease,suchasarteriosclerosis,hypertension,peripheralvasculardisease
anddiabetes. Alterationinendothelialfunctionprecedesthedevelopmentofmorphologicalchanges
inarterioscleroticdiseaseandcontributestoIMTdevelopmentandlaterclinicalcomplications[57].
Low levels of Nox activity are evident under normal physiological conditions. Upregulation of
the various Nox isoforms is associated with vascular pathology in response to injury underlying
arteriosclerosis, diabetes, obesity, hypertension, and hypoxia [55]. In this regard, ECs express
fourNoxisoformsincludingthesuperoxide-generatingenzymesNox1,2,and5andthehydrogen
peroxide-generatingenzymeNox4[36]. Underphysiologicalconditionstheseenzymesareexpressed
atrelativelylowlevelsonmembranesoftheendoplasmicreticulum(ER)andnucleuswherethey
contribute to intracellular redox signalling processes. However, in cardiovascular disease such as
atherosclerosis,endothelialexpressionofNox2,andtoalesserextentofNox1,appearstobeenhanced,
resultinginexcessivesuperoxidegeneration—especiallyintheextracellularcompartment[36].
Recent studies on human arteries from patients with coronary artery disease (CAD) [58],
and experimentally induced hypertension [40], diabetes and atherosclerosis [59] in animal model
systemssuggestthatNox1,2and5allpromoteendothelialdysfunction,inflammation,andapoptosis
withinthevesselwall[1],whereasNox4isbycontrastvasoprotectiveviaincreasingNObioavailability
andsuppressingapoptoticpathways(Figure2). Itisclearfrommostinvitrostudiesthatthefunctional
roleofNox4isbeneficial[60],inparticular,bymediatinganendothelium-derivedhyperpolarizing
factor(EDHF)-typeendothelium-dependentvasodilatationtosystemicallylowerbloodpressure[61].
Endothelial-specificoverexpressionofNox2generatesO − andpromotesvasculardysfunction
2
andoxidativestress[40]. SimilareffectsarereportedfollowingectopicexpressionofNox1within
vSMCs [62]. Hence, it is thought that Nox-derived O − reacts with endothelium-derived NO to
2
loweritsbioavailabilityandincreasethegenerationofONOO−. ThefreeradicalONOO− ishighly
reactiveandoxidizescysteinesandtetrahydrobiopterin,anessentialcofactoroftheeNOS.Lackof
tetrahydrobiopterin uncouples the enzyme, and promotes further O − generation exacerbating
2
the dysfunction [63]. Unlike O −, Nox4 derived H O doesn’t react with NO but may induce
2 2 2
NOsynthase[64].
ItisalsoclearthattypeofROSandthesiteofproductionisofparticularimportance. Despite
generating H O , ectopic expression of endothelial Nox4 did not promote medial hypertrophy in
2 2
contrasttoectopicexpressionofNox4invSMCordirectapplicationofH O [61]. TheH O may
2 2 2 2
counteractthehypertrophyorreactwithperoxiredoxins,whichsubsequentlymediatesignallingby
thiol-basedredoxreactions[65].
AlthoughpreclinicalstudieshaveprovidedcompellingevidenceforselectivetargetingofNox1
andNox2inECs,itremainsunclearwhetherchronicinhibitionofNoxactivityinhumanswillalsobe
efficacious. Oneinhibitor,apocynin,isthoughttoinhibitNoxactivitybyblockingphosphorylationof
p47phox,andconsequentlyitsinteractionwithmembrane-boundoxidasecomponents[66].Short-term
administrationofapocynintohumansiswelltoleratedandeffectiveatreducingROSproduction,
butitisunclearwhetherthepharmacodynamicandpharmacokineticprofilesofapocyninaresuited
tolong-termadministration[67].
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Figure 2. Simplified schematic of common forms of Nox enzymes in endothelial cells. H O can
2 2
beconvertedtoH Obycatalase(CAT)orglutathioneperoxidase(GPx). O − canalsoreactwith
2 2
NO to generate the reactive nitrogen species ONOO− that could be converted into NO ·, which
2
reactswithproteintyrosineresiduestogenerateNO -Tyr. Thisreactioncanleadtoadecreasein
2
thebioavailabilityofNO,leadingtoendothelialdysfunction. Nox-derivedROSmayalsocontrol
endothelial-mesenchymalstemcelltransition(EndoMT)[36].
4. NoxandVascularSmoothMuscleCells
The role of Nox isoforms within the medial layer has also been addressed. A hallmark of
arterioscleroticdiseaseistheaccumulationofvSMC-likecellsresultinginIMT[9].Severalstudieshave
focussedontheputativerolesofdifferentNoxisoformsinvSMCthatgenerateROSandthesubsequent
repercussions on vSMC cell phenotype and fate that contribute to vascular disease progression
(Figure 3). The intracellular signalling pathways responsible for ROS generation are still unclear,
but it is known that Nox1, 2, 4, and 5 are all expressed in vSMC but may differ in their activity,
responsetostimuli,andthetypeofROSreleased[68].
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Figure3. SimplifiedschematicofcommonformsofNoxenzymesinvascularsmoothmusclecell
(vSMCs).Nox4,onceactivated,generatesH O .Nox1expressionisincreasedearlyanddecreasedwith
2 2
lesionprogression,whileinductionofNox4isalateevent.Nox2andp22phoxareelevatedthroughout
lesiondevelopment.SMCshaveincreasedgenerationofROS,cellcyclearrest,evidenceofsenescence,
andincreasedsusceptibilitytoapoptosis[69].
Several recent studies have provided exciting new insight into the molecular mechanisms
by which agonists, through Nox1 generation of ROS, may promote vascular lesion formation.
Vascular SMC migration is pivotal to lesion formation and IMT and is considered an integrated,
dynamic, and cyclical process dependent upon orchestrated control of regulators of the actin
cytoskeleton,includingcofilin. CofilinisamajoreffectorofNox1-mediatedvSMCmigrationinvitro
and is notably reduced in Nox1-deficient (Nox1−/−) cells under both basal and PDGF-stimulated
conditions [70]. Thrombin-induced shedding of N-cadherin and vSMC migration is mediated by
Nox1-dependent transactivation of the epidermal growth factor receptor (EGFR) and subsequent
activationofmatrixmetalloproteinase-9(MMP-9). Nox1-dependentROSgenerationwithinvSMC
involves (i) Nox1-dependent transactivation of EGFR via c-Src; (ii) transactivation of EGFR
leadingtoextracellularsignal–regulatedkinases(ERK1/2)phosphorylation; (iii)Nox1-dependent
EGFR transactivation leading to MMP-9 activation via ERK1/2 phosphorylation, and (iv)
Nox1/EGFR/MMP-9-inducedN-cadherinshedding[71].
ThemechanismbywhichNox1promotesmigrationviaN-cadherinsheddingandtheroleof
N-cadherin in modulating SMC migration remain controversial, other, yet undefined, pathways
may also be involved. It is unclear whether a diminished cofilin/MMP-9 axis contributes to the
dysfunctional migratory response of Nox1-deficient cells in response to thrombin [71] or whether
thrombin induced ROS engages a Slingshot-1L (SSH-1L)-cofilin pathway to regulate cytoskeletal
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organizationandvSMCmigrationwithinvascularlesions. Nox1-mediatedstimulationofMMP-9may
alsoberesponsibleforincreasedplasmaclusterofdifferentiation-44(CD44)levelsinapoE−/− mice
followingproteolyticcleavageofmembrane-anchoredCD44[72]. Whilebasicfibroblastgrowthfactor
(bFGF)-andPDGF-induced,Nox1-dependentmigrationofvSMCisdependentonc-Jun-N-terminal
kinase (JNK) and Src/phosphoinositide-dependent kinase-1 (PDK1)/p21-activated protein kinase
(PAK),respectively,thrombin-inducedvSMCmigrationappearsdependentonROS-regulatedp38
MAPKactivation[73]. APoldip2mechanismforvSMCmigrationviaregulationoffocaladhesion
turnover and traction force generation in a Nox4/RhoA/focal adhesion kinase (FAK)-dependent
manner has also recently been reported [74]. While many of the aforementioned studies on Nox
isoformswereperformedusingvSMCinculture,thelikelihoodthatthesecellsarede-differentiated
vSMCs[75]and/orstemcellderivedmyogenicprogeny[18]and,thus,maynotrepresenttheputative
responseofdifferentiatedvSMCstochangesinNoxactivitycannotberuledout. Itremainstobe
seen whether these mechanisms observed invitro are operational invivo under physiological or
pathophysiologicalconditions. Ofnote,previousstudieshavereportednochangesinSrc,p38MAPK,
orJNKinresponsetoPDGFinwild-typeorNox1-deficientcells[69].
Asarterialinjuryandarterioscleroticlesionsarecharacterizedbyenhancedthrombinexpression
andactivitywithincreasedNoxactivityandROSproduction[72],activationofthissignallingcascade
bythrombinidentifiesonepotentialmechanismbywhichNox1-derivedROSmaypromoteIMT.
Enhanced Nox-1 expression is also associated with elevated O − levels within intimal and
2
medial vSMCs following carotid vascular injury [76]. Nox-derived ROS may also participate in
neointimalformationbyPDGF-inducedsignalling[77]. Indeed,ROSproducedbyNox5mayplay
animportantroleinPDGF-inducedJAK/SignalTransducerandActivatorofTranscription(STAT)
activationandvSMCproliferation[78]. PDGFisheavilydependentonJAK/STATactivationsince
specific knockdown of Nox5 reduces PDGF-induced ROS production and proliferation of these
cell and inhibits PDGF-stimulated JAK2 and STAT3 phosphorylation [78]. Other studies have
implicatedNox1invSMCphenotypicchanges,includingangiotensinII-inducedhypertrophy[79],
serum-inducedproliferation[80],andbFGF-inducedmigration[81]andstrain-inducedphenotypic
switching[82].VascularSMCspecificdeletionofNox1reducedIMTinresponsetofemoralarterywire
injury[69]whileectopicexpressionofNox1exhibitedincreasedPDGF-inducedO − productionin
2
aortic vSMCs concomitant with increased growth and migration [69] . EGF, FGF-2, angiotensin II
(AngII)andtheplasminogen/plasminsystem(uPA)allpromoteNoxactivitywithvSMCinvitroand
contributetoIMTinvivo[69]. Finally,theprecisemechanismofhowNoxmediatedROSdeteriorates
vascularfunctionandpromotesvascularremodellingandIMTinvivohasbeenrecentlyelucidated.
CyclophilinA(CyPA),a20kDchaperoneproteinthatissecretedfromvSMCsinresponsetoROSand
stimulatesvSMCproliferationandinflammatorycellmigrationinvitroandinvivo[79].
DespitethebeneficialsignallingroleofNox4generatedH O fromtheendothelium,excessive
2 2
concentrationsfromvSMCmayinduceinflammation,fibrosis,apoptosis,andevennecrosis[83].Given
theconstitutivenatureofNox4activity,theregulationofH O formationmaybeharmfulorprotective
2 2
depending on the levels and source of ROS generated [60]. Under certain conditions, such as the
releaseoftransforminggrowthfactor-β1,diabetes,andheartfailure,Nox4-dependentH O formation
2 2
maybecomeharmful.
5. TheRoleofNoxinAdventitialCells
The role of Nox isoforms within the adventitial layer has also been addressed (Figure 4).
Arteriosclerotic vessels are characterized not only by IMT, but also increased stiffness secondary
to collagen and elastin deposition, a process regulated by the adventitial fibroblast and termed
fibrosis. Thefibroblastistheprimarycelltypeoftheadventitiallayerandcontributestothecontinual
reorganization of the extracellular matrix via matrix deposition and secretion of growth factors,
chemokines,andinflammatorycytokines[84].Fibroblastsalsoinfluenceandpromotetheinflammatory
responsebyfacilitatingleukocyterecruitment,survivalandfunction. Theimportanceofthevascular
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adventitia to IMT and arteriosclerotic disease progression has also recently been highlighted [85].
Compellingevidencenowsuggeststhatthevascularadventitiaisactivatedearlyfollowingvascular
injuryandduringIMTprogressionandmayplayanimportantroleinvascularinflammationassociated
witharterioscleroticdisease[86]. AdventitialcellsproducealargeamountofNox-derivedROSin
responsetovascularinjurythatcanleadtoanexpandedvascularisationthroughthevasavasorumand
deliveryofinflammatorycellstotheadventitiaandoutermediaculminatinginvascularhypertrophy
andhyperplasia[87].
Figure 4. Simplified schematic of common forms of Nox enzymes in adventitial cells. Nox2 and
p22phox are predominant in the adventitia of hypertensive vessels and promote the secretion
of monocyte chemoattractant protein-1 (MCP-1) and interleukin 6 (IL-6). Nox1 and Nox4 with
Nox4-derivedH O consideredprotectivearealsopresent[87].
2 2
Earlystudiesrevealedabundantco-localizationofNox2,withmembranep22phoxandcytosolic
p47phox and p67phox in aortic vascular adventitia and enrichment of p67phox-dependent Nox
activityinculturedadventitialfibroblasts[88]. AdventitialcellsalsoexpressNox1andNox4with
Nox4-derivedH O consideredprotective. However,Nox2-derivedROSremainsoneofthemain
2 2
isoformforadventitialROSsignalingwithadventitialleukocytesexhibitingsignificantexpression[37].
Nox-2isenhancedbyseveralstimuliincludinghypoxia,cytokines,hormones,metabolicfactorsand
mechanicalinjury[87].
Adventitial Nox2 generated ROS also plays an important role in angiotensin II-hypertension
associated IMT by modulating the secretion of monocyte chemoattractant protein-1 (MCP-1) and
interleukin6(IL-6)[89].AdventitialNox2isactivatedinhypertensionmodels[90]whileROSsignaling
alsopromotesvSMChypertrophyviaaquaporin1andNox1[91]. Thesestudiessuggestaputativerole
foradventitia-derivedROSinmedialSMChypertrophyandneointimalhyperplasia. Thesuperoxide
anionmetabolite,H O isthelikelyresponsiblefortheadventitia-derivedparacrinesignallingacross
2 2
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thevesselwallasitishighlystable,cellpermeant,andcapableofactivatingdownstreamsignaling
mechanismsinvSMCs,leadingtophenotypicmodulationandde-differentiation[86].
Itiswidelyacceptedthatthefibroblast/myofibroblastisthecellmostresponsibleforinterstitial
matrixaccumulationandconsequentstructuraldeformationsassociatedwithadventitialfibrosis[92].
AccumulatingevidencenowsuggeststhatoxidativestressresultinginROSgeneration,mainlyinthe
formofsuperoxideandhydrogenperoxide,playsasignificantroleintheinitiationandprogression
ofadventitialfibrosis[93]. Moreover,Nox4mediatedROS-inducedfibroblastcellactivationiskey
eventwhereitfacilitatesTGF-β1signallingoffibroblastactivationanddifferentiationintoaprofibrotic
myofibroblastphenotypeandmatrixproduction[94].
6. TheRoleofNoxinStemCells
Resident stem/progenitor cells are characterized by their unique ability to self-renew and
proliferatewhileretainingtheirabilitytodifferentiateintospecializedcellswithinthebody. Thereare
severalreportedresidentvascularprogenitorcellsresidinginallthreelayersofthevessel;theintima,
media and adventitia, each displaying the ability to differentiate into vascular cell lineages [15].
Theseprogenitorstemcellpopulationsaremultipotentandhavethecapabilityofdifferentiatinginto
MSC-likecells[12]. ResidentvascularMSCs-likecellsarepericytesdefinedasmultipotentstromalcells
withtheabilitytodifferentiateintocellsassociatedwithmesodermalandneuroectodermallineage
includingvSMCs,osteoblasts,adipocytesandchondrocytes[95].
ROS play an important role in dictating the fate of normal stem cells. Low levels of ROS
are required for stem cells to maintain quiescence and self-renewal whereas increases in ROS
productionareassociatedwithstemcellproliferation/differentiation,senescence,andapoptosis[96].
The production of ROS in stem cells is therefore by necessity tightly regulated to maintain tissue
homeostasisandrepairbyvariousintrinsicandextrinsicfactors,whichmaybecomealteredleadingto
dysregulationofROSproductionundervariouspathologicalconditions. ThepresenceofNoxinstem
cellsisthoughttohaveafunctionalroleasO sensorand/orasalow-levelROSproducerandredox
2
messengerforcontrollingcellgrowthanddifferentiation[25].
In order to better understand the differentiation process, several studies have focused on the
metabolicchangesthatoccurduringdifferentiation[97,98]. Inanundifferentiatedstate,MSCreside
inaspecialmicroenvironmentcalledthestemcellniche. Thepurposeofthis“niche”istokeepcells
inanundifferentiated,quiescentstatemaintainingtheirstemness. Forthistohappen,O levelsare
2
keptataminimum. WhenO levelsarekeptlow,thestemcellnicheisforcedtoturntotheuseof
2
analternativeanaerobicmetabolismforenergy[99,100]. This,inturn,resultsinlowlevelsofROS
productionduetothelackofavailableoxygenkeepingproliferationactivityataminimumwhilst
maintainingthecellscorestemcellpropertiesofself-renewalanddifferentiationabilities. During
thetransitionofstemcellsfromtheirundifferentiatedstatewithintheniche,severalchangesoccur.
ThenumberofmitochondriapresentinthecellincreasesinresponsetoO levelsleadingtoincreased
2
levelsofROSproduction. Althoughthisgeneralprocessispresentacrossalldifferentiationcells,MSCs
displayauniqueredoxpathwaydependingontheirfatesuggestingthatMSCshaveexplicitredox
profilesdependingontheirultimatelineagespecificity[101].
ThemainsourceofROSinstemcellsisthemitochondria.Thisoxidativereductionprocessistriggered
byaleakageofasmallnumberofelectronsfromtheelectrontransportchain.Thesemitochondrialelectrons
reactwithmolecularO resultinginformationofO −,aprecursorforROSgenerationwithinthestem
2 2
cell[102].Theoxidativereductionprocessissuccessfullyachievedthroughtheactivityofseveralvital
mitochondrialrespiratorycomplexformationsontheinnermitochondrialmembranespace. Theinitial
complex I formation is composed of Nox enzymes and acts as a catalyst during the oxidation of
NADPHtoNADP+leadingtotheformationofsuperoxideradicals[102]. ThemainNoxenzymesin
MSCsderivedfromadiposetissueresponsibleforthisprocessareNox1and4[103]. Othersourcesof
O − maybegeneratedatvaryinglevelsinundifferentiatedhumanMSCsbycomplexIIcomprising
2
of succinate dehydrogenases [99]. The enzyme works by catalysing the oxidation of succinate to