Unveiling LOX-1 receptor interplay with nanotopography: mechanotransduction and atherosclerosis onset.

1.2 MB·English

by  Di Rienzo, Carmine

Unveiling LOX-1 receptor interplay with
nanotopography: mechanotransduction
SUBJECTAREAS: and atherosclerosis onset
NANOSCALEBIOPHYSICS
BIOMATERIALS-CELLS
CarmineDiRienzo1,2,EmanuelaJacchetti1,FrancescoCardarelli2,RanieriBizzarri1,
TISSUEENGINEERINGAND
REGENERATIVEMEDICINE FabioBeltram1&MarcoCecchini1
IMPLANTS
1NEST,IstitutoNanoscienze-CNRandScuolaNormaleSuperiore;PiazzaSanSilvestro12,56127Pisa(ITALY),2Centerfor
NanotechnologyInnovation@NEST,IstitutoItalianodiTecnologia,PiazzaSanSilvestro12,I-56127Pisa,Italy.
Received
3December2012
Lectin-likeox-LDLreceptors(LOX-1)playacrucialroleintheox-LDL–inducedpathological
Accepted
transformationofvessel-wallcomponents,acrucialearlystepinatherogenesis.LOX-1dynamicsis
17December2012
quantitativelyinvestigatedinhumanendothelialcells(HUVECs)exposedtoenvironmental
nanotopographies.Wedemonstratedistinctnanotopography-inducedcellphenotypes,characterizedby
Published
differentmorphology,LOX-1diffusivityandoligomerizationstate:HUVECsonflatsurfacesexhibitthe
25January2013
behaviorfoundinpro-atherogenicconditions,whilegrowthonnanogratingscaninterferewithLOX-1
dynamicsandleadtoabehaviorcharacteristicofnormal,non-pathologicalconditions.
Correspondenceand
requestsformaterials Atherosclerosisoccursinlargeandmedium-sizearterieswherelesionscontaininglipids,proteinanddeath
cells can form1. The appearance of atherosclerotic plaques in defined spatial regions suggests a local
shouldbeaddressedto
inclinationtowardspro-atheroscleroticpathwaysconcomitanttoadiffuseactivationofanti-atheroscler-
M.C.(marco.
oticpathways2.Oxidizedlowdensitylipoproteins(ox-LDLs)areconsideredamongthemosteffectiveactivators
[email protected]) ofthepro-atheroscleroticpathwaysandwerelinkedtoatherogenesisandplaquerupture1,3.Inthiscontext,the
orR.B.(r.bizzarri@sns. endotheliallectin-likeox-LDLreceptor(LOX-1)playsacrucialroleintheox-LDL–inducedpathologicaltrans-
it) formationofvessel-wallcomponents,acrucialearlystepinatherogenesis4,5.Anumberofinvestigationshave
linkedLOX-1toatherogenesis.Itwasshown,forinstance,thatmicelackingLOX-1againstabackgroundofnull
LDLreceptorsexhibitsignificantlyreducedatheroscleroticplaqueformationcomparedtomiceharboringLOX-1
insimilarbackgroundconditions6.Bycontrast,miceoverexpressingLOX-1againstanapolipoproteinE(apoE)-
deficientbackgroundexhibitanenhancedatheroscleroticplaqueformation7.Theseobservationsindicatethat
ox-LDLbindingtoLOX-1acceleratesformationandprogressionofatheroscleroticplaques.
Fromamolecularpointofview,itisworthnotingthatwell-knownchemicalstimuliinvolvedinatherogenesis
arealsoabletoregulateLOX-1expressiononthecellmembrane8–13.Inthiscontext,ithasbeenrecentlyreported
thattheLOX-1dimerisnecessaryforefficientox-LDLuptake.Furthermore,thedimerizationprocessiscon-
centration-dependentanditdoesnotneedox-LDLstimulation14–16.Thus,LOX-1isexpressedatlowconcen-
trationandkeptmonomericinanormalendothelium,whileitisoverexpressedandstartstoaggregateduring
theearlystagesofatherosclerosis17,18.Therecurrentdispositionofatheroscleroticplaques(i.e.closetoarterial
bifurcations) suggests that their formation results from a highly regulated interplay between homogenously
distributedchemicalstimuli(e.g.ox-LDL)andlocalizedpro-atheroscleroticevents,asithasbeendemonstrated
in detail for physical stresses19. Here, cytoskeleton-mediated and integrins-mediated mechanotransduction
clearlyemergesasoneofthemaindrivingforcesinendothelialdysfunctionattheearlieststepsinatherogen-
esis2,20,21.Theeffectivenessofmechanotransductiondependsonseveralexternalregulators,includingshearstress,
cell-cellinteractions,andcell-matrixinteractions19,22–25
Inspiteofthesefindings,noonehasyetadequatelydescribedthecorrelationbetweentheextracellularmatrix
topographic features and atherogenesis onset. Indeed, ECM topography stands as one of the most relevant
modulators of cell physiology and provides a ‘‘non-chemical’’ activation pathway to tune cell functionality/
properties26–28. Accordingly, in this work artificial scaffolds with controlled nano-/micro-topography were
appliedtoinvestigatehowcell-ECMinteractionsinfluenceLOX-1behaviour.Morespecifically,wereporton
LOX-1receptordynamicsandaggregationonthecellmembraneanditsinteractionwiththecytoskeletonandthe
extracellularenvironmentindifferenttopographiccontexts.NotablyweusedaGFP-taggedvariantofLOX-129
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along a preferential axis that follows the substrate directionality
(Fig. 1 b and Suppl. Fig. 2 a–b). In order to quantify the polari-
zation on the different employed geometries (Suppl. Fig. 1), we
measured cell spreading (adhesion area), cell elongation (L) and
cell alignment to substrate directionality (Q) (Fig. 2 and Suppl.
Fig. 3). The mean adhesion area of HUVECs on FLAT substrates
is 1600 6 200 mm2, in good agreement with literature data37.
Conversely, HUVECs covered T1 samples with a significantly
reduced area compared to the FLAT surfaces (40%, Suppl. Fig. 3
a). Significantly, on longer-period topographies (T2, T4) cells
showed spreading areas similar to FLAT surfaces (Fig. 2 and
Suppl. Fig. 2 a). As previously stated, HUVECs showed increased
elongationalongthepatterndirectiononT1,T2,andT4,compared
to FLAT values (Suppl. Fig. 3 b and c). Maximum elongation was
detectedontheT2substrates(L52.260.2comparedtoL51.06
0.1onFLAT,Fig.2b)togetherwithoptimalalignmenttosubstrate
directionality(Q510.0u60.5u,Fig.2c).Takentogether,thesedata
demonstratethatT2performedbestduringHUVECspreadingand
contactguidance.Forthisreason,weselectedT2forthefollowing
experiments,andkepttheFLATasananisotropiccontrolcondition.
Wetestedhownon-physiologicalconditionsassociatedwithspecific
cytoskeletaldegradationaffectthemorphologyofHUVECsattached
toflatorpatternedsubstrates.Firstweinvestigatedtheeffectofmicro-
tubuledisruptionbyadministratingnocodazole(NCZ).OnT2NCZ-
Figure1|EffectoftopographyonHUVECmorphologyandLOX-1 treated cells showed a reduction in alignment from Q510.0u60.5u
spatialdistribution. RepresentativebrightfieldimagesofHUVECs (non-treatedcells)toQ516u62u(Suppl.Fig.4c),and,inelongation,
adheredon(a)FLATand(b)nanogratings.RepresentativeTIRFMimages from L52.260.2 (non-treated cells) to L51.160.1 (Suppl. Fig. 4 b).
ofLOX-1-GFPtransfectedHUVECson(c)FLATand(d)nanogratings. Notably,NCZ-treatedcellsshowedareducedelongationfactorwithall
Scalebar10mm.Whitearrowsindicatethesubstratedirectionality. substrates(Suppl.Fig.4b).Then,wetestedtheeffectofactin-filament
disruptiononpolarization.CytochalasinD-treatedcellsshowedneg-
ligible variations of elongation and alignment, although a detectable
that can be transiently transfected into human endothelial cells reduction (about 40%) of the adhesion area on both T2 and FLAT
(HUVECs)andisnotactivatedbyexternalligands,thusproviding substrates was observed (Suppl.Fig. 3a). Finally,we tested the con-
the ideal model to study the sole influence of cytoskeletal factors tractility depletion effect by treating HUVECs with blebbistatin.
andexternaltopographyonthereceptormobilityandaggregation. Treated cells showed no significant difference in spreading area on
Receptor dynamics was quantitatively investigated by combining FLAT or T2 (Fig. 2 (a)). On the other hand, we measured strongly
high-resolution fluorescence microscopy, fluorescence correlation reducedelongationonT2surfacecomparedtothephysiologicalstate
techniques, and micro-/nano-fabrication methodologies. First, we (Fig.2(b))whichledtovaluescomparabletothecellelongationon
testedtheeffectsofmechano-topographicalstimulionLOX-1diffu- FLAT.Atthesametimeweobservedreducedalignmentbetweenthe
sivity in cells using an original approach that combines advanced cellsandthepattern(Fig.2(c)).
fluorescence correlation techniques30,31 and micro-/nano-fabrica-
tion. Next, we investigated the relation between cellular function/ LOX-1-GFPlocalizationanddiffusivity.Inafurthersetofexperi-
structureandLOX-1diffusivitybytreatingcellswithcytoskeleton- ments, we investigated how adhesion on FLAT or T2 modulates
disruptingagents.Finally,welinkedourresultsonLOX-1diffusion localization and single-molecule mobility of LOX-1-GFP. Single-
invarioustopographicconditionstoitsoligomerizationstateinliv- moleculemobilitywasassessedbyfluorescencecorrelationspectro-
ingcellsbasedonafluctuationspectroscopictechniquenamednum- scopy (FCS) measurements. Average membrane mobility of the
berandbrightness(N&B)analysis32.Ourresultsclearlydemonstrate receptor in each point was determined by correlating images of a
the existence of distinct topography-induced endothelial pheno- temporal stack, according to the time image correlation technique
types,characterizedbydifferentcellmorphology,reactive-oxygen- (tICS)31.Line-scanningFCSwasappliedtodeterminethedirectional
specie (ROS) production, LOX-1 diffusivity and oligomerization mobility of the receptor between two given points in a segment30.
state.Mostimportantly,thedataallowustoidentifypeculiarpro-/ Additionally, the extent of autocorrelation at each point along the
anti-atheroscleroticconditionsthatmighttakeplaceduringathero- lines allowed estimating the relative concentration values, since
sclerosisonset. correlation amplitude and molecular concentration are inversely
proportional38.
Results Figure1canddshowsrepresentativeimagesofHUVECstran-
MorphologyofHUVECsonnanostructuredsurfaces.Inthefirst siently transfected withGFP-tagged LOX-1 onFLAT and T2 sub-
setofexperiments,westudiedthemorphologyofHUVECsonFLAT strates,respectively.LOX-1-GFPdistributionisuniformonFLAT.
and patterned surfaces. In order to determine suitable substrate By contrast, a non-uniform membrane distribution can be clearly
geometry for efficient HUVEC mechanotransduction, we initially seenonthepatternedsubstrates(furtherevidenceofthiseffectisalso
assayednanogratingsofthreedifferentperiods,namely1mm(T1), reportedinSuppl.Fig.2forothergeometries).Inparticular,‘ridge’
2mm(T2)and4mm(T4)(seeMaterialsandMethodsfordefinitions regionsarealwayscharacterizedbysignificantlyhigherfluorescence
andcharacteristics).Figure1andSuppl.Fig.2providerepresentative intensity compared to ‘groove’ regions, as detected by TIRF and
imagesofHUVECs attachedonFLAT, T1,T2andT4.OnFLAT, confocalimaging.Line-scanningFCSallowedattributingthehigher
cells were found to spread without polarization and showed the fluorescence to higher LOX-1-GFP concentration on the ridges
typical star-like shape (Fig. 1 a). By contrast, on patterned sub- (Suppl. Fig. 3). Indeed, correlation amplitudes in grooves were
strates they showed polarized morphology, with the cell stretched alwayshigherthanontheridges.
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Next, we measured average LOX-1-GFP diffusivity by tICS. By pCFcalculatedatthedistanceof1mmparalleltothesubstratedirec-
fittingtICSdatatoa2Dmodelofdiffusionsuitableformembrane tionality(redcurve,Fig.3b)yieldsacorrelationpeakearlierintime
studies39,40, it was found that D50.08160.004 mm2/s on FLAT withrespecttothatcalculatedorthogonally(bluecurve,Fig.3b).This
(Fig.2(d)),avalueinagreementwiththosedisplayedbymembrane indicates that LOX-1-GFP moves faster along ridges and grooves
proteins of similar size41,42. Remarkably, on T2 we measured an than across them. This effect was quantified by the mobility ratio
almosttwofoldincreaseinthediffusivityofLOX-1-GFP[D50.143 R(ratiobetweenthetimeofCFpeaksrelevanttoparallelandortho-
60.006 mm2/s, Fig. 2(d)]. In order to strengthen and support our gonaldirectionstopattern,respectively).ForT2,R51.860.8,indi-
analysis, we also performed the same experiment on T1 and T4, catingasignificantanisotropyinLOX-1-GFPmobilityonpatterned
obtainingsimilarvalues(Suppl.Fig.3d). substrates. As a control, the same analysis was repeated on FLAT
UponNCZtreatment,wefoundD50.12460.006mm2/sonT2, (Fig. 3c):thepCFscalculated intwoorthogonal directionsyielded
andD50.08260.004mm2/sonFLAT(Suppl.Fig.3d).Thisslight correlation maxima with no clear difference in their timing. This
reduction of diffusivity on T2 substrates compared to the physio- suggeststhatthereisnopreferentialroutetakenbyLOX-1-GFPin
logicalstatehasnohighstatisticalsignificance.Thesametrendwas HUVECsadheredonFLAT.Accordingly,wefoundameanmobility
observeduponCytochalasinDtreatment(D50.12460.007mm2/s ratioR51.060.3forFLAT.ThisresultreinforcestheideathatLOX-
onT2,andD50.0860.01mm2/sonFLAT,Suppl.Fig.3d).Thus,the 1-GFPmobilityissignificantlyinfluencedbythespatialdistribution
membranemobilityofLOX-1-GFP wasfoundtobeunaffectedby ofridgesandgroovesthatarepresentinthepatternedsurfaces.
microtubule and actin depolymerization both on FLAT and pat-
ternedsurfaces.Conversely,themobilityofLOX-1-GFPonT2was LOX-1-GFP aggregation state in living cells. It is by now widely
significantlyhampered(D50.1060.01mm2/s,Fig.2d)byblebbis- accepted that LOX-1 homodimerization is needed for ox-LDL
tatin and became statistically indistinguishable from FLAT values internalization in living cells14, and that its oligomerization state
(D50.1060.02mm2/s,Fig.2d).Thissuggestsakeyroleofcontract- may be locally regulated by receptor density15. Thedata presented
ility in the topography-induced modulation of LOX-1-GFP so far pointed out detectable differences between LOX-1-GFP
mobility. behavior on FLAT as compared with nanogratings. To investigate
Inordertofurtherinvestigatetheinfluenceofpatternedsubstrates whetherthismayberelatedtoitsoligomerizationstateweperformed
on the membrane mobility of LOX-1-GFP, we adopted the pair the number and brightness analysis (N&B)32 on stacks of fluore-
correlationfunction(pCF)approachtolineFCS30,43.Indeed,byana- scence images. The results obtained are displayed in Fig. 4. As a
lyzing the spatial correlation of fluorescence fluctuations at a pair reference for ‘monomeric’ molecular brightness on the membrane
ofpointsineachsampleseparatedbyadistancecomparabletothe we transiently transfected HUVECs with a farnesyl-conjugated
topographyperiod,wewereabletoprobethesingle-moleculemobil- GFP39 (Fig. 4 a, central panel). As expected we found a homo-
ityanddirectionalityofLOX-1-GFPmembranetrafficwithrespect geneous distribution of the monomer on the membrane (Fig. 4 a,
tothesubstratestructure.Allexperimentswerecarriedoutatphysio- left panel, points in the green rectangle) with a corresponding
logicalconditions:arepresentativepCFmeasurementisreportedin average molecular brightness (e ) of 0.06660.003 counts/
monomer
Fig. 3. For simplicity, we focused on pair correlations along two molecule/pixel-dwell-time. This value was taken as reference for
directions: perpendicular (blue arrow, Fig. 3a) and parallel (red analogous measurements on cells transiently transfected with
arrow, Fig. 3a) to the substrate pattern directionality. For T2, the LOX-1-GFPonFLATandT2substrates(Fig.4e–fand4h–i).
Figure2|EffectofcontractilityinhibitiononHUVECsMorphologyandLOX-1diffusivityonFLATandT2substrates. Thehistogramsreport
(a)theadhesionarea,(b)elongationand(c)alignmentofLOX-1transfectedcells,and(d)LOX-1diffusivity.Experimentswereperformedwithand
withoutblebbistatinadministration.Dataarereportedasmean6standarderrorof4experiments.275cellswereanalyzed.Significantdifferences
betweenthepopulationmeansarereported.
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Figure3|AnisotropicLOX-1diffusion. (a)SchemeofLOX-1-GFP-transit-timemeasurewheretEistheLOX-1meantimetocross1mmalong
thepatterndirectionandt\isthesamecoefficientalongtheorthogonalone.(b–c)RepresentativeLOX-1pCFalongthetwodirections(parallelred
curve,perpendicularbluecurve)ofasingleHUVECcelladheredon(b)T2and(c)FLATsubstrates.Theinsetsrepresentthetwoanalyzeddirections.
Inlinewiththeliterature,N&BanalysisofLOX-1-GFPonFLAT endothelial activation, dysfunction and injury46. Recently, a novel
substratesshowsacoexistenceofmonomersanddimers,resultingin lectin-like scavenger receptor for ox-LDL (LOX-1) was identified,
a mean molecular brightness of 0.10260.008, approximately 1.5 primarilyintheendothelialvascularsmoothmuscle,lymphoidcells
timeshigherthanthemonomericreferencevalue.Thisresultsug- (including macrophages) andplatelets, whichallows theuptake of
gests that LOX-1-GFP transfection yields membrane protein con- ox-LDL and cell activation4,47–49. Also, recent transgenic animal
centration values above the dimerization threshold (Fig.4 e–f). model studies show that LOX-1 plays a significant role in athero-
Remarkably,asimilaranalysisdemonstratesthatthedimericpopu- sclerotic plaque initiation and progression6,7,50. Administration of
lation almost completely disappears when cells are plated on T2 LOX-1 antibodies in cellular and animal models consistently sug-
substrates,showingaconcomitantincreaseofthemonomericform gests that such intervention inhibits atherosclerosis51. Despite its
fraction(e50.07060.004,Fig.4g–i). important role in the process of atherogenesis, the most effective
means of targeting and disrupting OxLDL-LOX-1 endocytosis
Reactive oxygen specie (ROS) production of HUVECs remains elusive. Promisingly, Ishigaki et al showed that LOX-1
on nanostructured surfaces. In order to study the effect of expressed ectopically in the liver via adenovirus administration
nanotopography on HUVEC oxidative stress, cytoplasmic ROS reducesthelevelsofcirculatingOxLDLandinhibitstheformation
concentration was measured for cells grown on T2 and FLAT. of atherosclerotic lesions52. Gene therapy with possible genomic
Using a combination of two dyes and fluorescence microscopy manipulationofscavengerreceptorexpressionbydeliveryoftrans-
(TBHP and c-H2DCFDA, see Materials and Methods for details), genesorbyblockadeofgeneexpressionwasalsosuggested53.Inour
oxidative stress could be quantitatively evaluated. Regardless of opinion,suchcomplexinvivoapproachescanbeusefullysupported
the presence of nanopatterning, our substrates did not induce byinvitrostudies,wheremanymoleculardetailsofLOX-1activity
significant stress in HUVECs. Indeed, ROS concentration on relevant to pathogenesis (e.g. diffusion, oligomerization state, etc.)
FLATwasreducedtoone-thirdwithrespecttothepositivecontrol canbeisolatedandstudiedindetailatthesingle-moleculelevel.To
inducedbyTBHP.Nonetheless,wefoundthatcellsonT2showeda thisaimweexpressedaGFP-labeledvariantofLOX-1inlivehuman
marked decrease (44610 %, p , 0.05, Mann–Whitney U test) in
endothelial cells and studied its behavior by means of high-
ROSproductionwithrespecttocellsontheFLAT.Thisindicatesthat
resolution/highsensitivityfluorescencemicroscopy.Thisvariantof
thepresenceofnanopatterningcanleadtoalessstressfulcondition
LOX-1isunabletobindtoox-LDLalthoughitretainsallfeaturesof
forendothelialcellsthanstandardflatsurfaces.
nativeLOX-1anditisthereforetheidealsystemtovisualizethesole
effectofECMtopographyonLOX-1-dependentcelldysfunction.
Discussion Experimentally, a toolbox of correlation-based techniques was
Thereisstrongevidenceforaroleofoxidativestressinallstagesof used to quantitatively address LOX-1-GFP local diffusivity (by
atherosclerosis44,45. Oxidized low density lipoprotein (ox-LDL), a tICS),spatialheterogeneityofflow(bypair-correlation)andoligo-
markerofoxidativestress,ispresentintheplasmaandintheathero- merizationstate(byNumberandBrightnessanalysis).Atthesame
sclerotic arteries of patients with atherosclerosis. Ox-LDL leads to time, by micro-/nano-fabrication methodologies we prepared
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Figure4|LOX-1aggregationstate. (a–c)CalibrationofmonomericGFPbrightnessusingfarnesyl-GFP.(a)fluorescenceintensityimageofacell
expressingfarnesyl-GFPshowswidespreaddistributionoftheproteinonthemembrane(b)Selectionofpixelswithbrightnesscorrespondingtothe
monomers(green,B51.066,e50.066)andtothebackground(blue)(c)brightnessvsintensityplotshowingthe‘‘green’’and‘‘blue’’selections.
(d–f)RepresentativeN&BanalysisofaHUVECcellonFLATwithLOX-1initsdimericform.(d)intensityimage(e)Selectionofpixelswithbrightness
correspondingtothedimers(red,B51.12,e50.12)andtobackground(blue)(f)cumulativeresults:percentageofcellsshowingtheLOX-1monomer
(anddimer)onFLAT.(g–i)RepresentativeN&BanalysisofaHUVECcellonT2withLOX-1initsmonomericform(g)intensityimage(h)Selectionof
pixelswithbrightnesscorrespondingtothemonomers(green,B51.07,e50.07)andtothebackground(blue).Cumulativeresults:inthiscaseaboutthe
90%oftheanalyzedcellsindicatesthepresenceoftheLOX-1monomeronly.
controlled-patternsubstratesallowinginvestigatingdifferentenvir- indirectingLOX-1diffusioninmembranes.Accordingly,bothcon-
onmentalconditionsforcellattachment,spreading,andgrowth.By tractilitydepletionandflatsubstratesareabletoabolishtheobserved
thisintegratedapproachwedemonstratedtheinterplaybetweencell anisotropic increase in LOX-1 diffusivity. In this context, a recent
spreading/polarization,LOX-1dynamics/oligomerizationstate,and workontheCD36macrophagescavengerreceptordemonstratesthe
LOX-1interactionswiththecytoskeletonandwiththeextracellular existenceofdifferentdiffusivebehaviors,suchasisotropicdiffusion
environment. In particular, as schematized in Fig. 5, we identified andlineardiffusion,‘‘guided’’bycytoskeletonelements.Thelatter
two distinct conditions: i) when placed in isotropic environments motion, interestingly, confers a higher effective diffusivity to the
(cellsculturedonflatsurfaces)LOX-1isevenlysplitintoahomo- receptorandcorrelatestoitsfunctionalaggregationstate42.
dimericandmonomericform,itishomogenouslydistributedwithin It is well known that LOX-1 homodimerization is required for
themembranewhereinitdiffusesisotropically,and,ii)wheninan receptoractivationandsubsequentox-LDLinternalization14.Based
anisotropicenvironment(polarizedcellsadheredonnanogratings) on this and on the results discussed above, the endothelial cells
LOX-1showspreferentialaccumulationinregionsofhigheradhe- adhered on flat substrates can be considered more atherogenesis-
sion (ridge) and is preferentially locked in its monomeric form pronethenendothelialcellsadheredonnanogratings(e.g.T2)where
throughoutthemembrane.ThemonomericformofLOX-1displays LOX-1islockedinaninactiveform(Fig.6).Inourresults,cellswith
higher diffusivity values that are dependent on contractility and similar LOX-1 expression levels yield remarkable differences in
topography. It is worth mentioning that topography orientation LOX-1 aggregation/activation state as a function of the substrate
drives the main orientation of the cytoskeleton components. This used.Overall,theseresultssuggestthatitispossibletoregulatethe
inturnsuggeststhatsuchorderedcellularstructuresmayplayarole LOX-1oligomerizationstate(andthenitsdispositiontointernalize
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atherosclerosis55.Basaloxidativestresscouldbemodulatedbychem-
icalenvironmentbutalsobytheECMtopographyand,inparticular,
ourresultsshowthatT2nanogratingisabletoreducetheendothelial
celloxidativestress.Alongthisreasoning,thetwoidealconditions
tested here (periodic grating vs smooth surface) may be related,
respectively, to the non-atherosclerotic intima, where endothelial
cellsareexposedtoanorderedmeshofcollagenfibers,andtothe
aged/atherosclerotictissue,wherethesamecellsareinsteadexposed
toacontinuousdepositionofamorphoussubstances,suchaselastin
andcollagen56–58.
Webelievethattheseresultsshouldbetakenintoaccountforthe
rationalengineeringofstentsforcardiovascularsurgery.Amongthe
drawbacks limiting their more widespread use is the long-term
endothelial dysfunction and inability to adapt to growth58. Many
studies demonstrate that topographical features modulate surface
endothelialization and endothelial integration with fluxes37,59. Our
results contribute to this picture by isolating and investigating
the role of nanotopographical modulation on protein activity in
endothelialdysfunctionandatherosclerosis.
Methods
Substratefabrication.Nanogratings(alternatinglinesofgroovesandridgeswith
submicrometerlateraldimension)werefabricatedbythermalnanoimprint
lithography(NIL)aspreviouslydescribed33.Copolymer2-norborneneethylene
[cyclicolefincopolymer(COC)]foils(IBIDI.Martinsried,Germany)wasselectedas
thermoplasticmaterialbecauseoffavorablebiocompatibilityandopticalproperties34.
Theimprintingprocesscomprises5steps:(1)thesubstrateisplacedbetweenthe
moldandaglasscoverslipinsidetheNILsystem;(2)thetemperatureisraisedto
Figure5|ROSproductionindifferenttopographicalcondition. 150uC;(3)50-bar-pressureisappliedfor5min;(4)thesystemiscooledto70uC;
Representativefluorescenceimagesofcellstreatedwithc-H DCFDAon (5)finally,thepressureisreleased.TheimprintedfoilsareremovedfromtheNIL
2
FLAT(a)andT2(b),respectively.Cellcontoursareshownforclarity. system,detachedfrommoldandthenattachedbyusingsiliconeglue(RS
Components,Italy)tothebottomofhollowed35mmPetridishes.Weproduced
Thewhitearrowindicatessubstratedirectionality.Scalebar10mm.
nanogratingscharacterizedbyperiodsof1mm(T1),2mm(T2)and4mm(T4),and
(c)NormalizedaverageROSconcentrationinHUVECsonFLATandT2. depthof350nm.
651cellswereanalyzed.Dataarereportedasmean6standarderrorof Beforeeachexperiment,thesubstratesweresterilizedinethanolfor15min,and
3experiments. thenwashedtwiceinPBSandinculturemedium.
Cellculture,transfectionandtreatments.Low-passagehumanumbilicalvein
ox-LDL)manipulatingthecellularenvironment.GenerationofROS endothelialcells(HUVECs)weregrowninM200mediumsupplementedwith2%v/v
is inevitable for aerobic organisms54, but healthy cells maintain a fetalbovineserum(FBS),1mg/mlhydrocortisone,10ng/mlhumanepidermalgrowth
factor(EGF),3ng/mlbasicfibroblastgrowthfactor(FGF),and10mg/mlheparin
controlled rate of ROS levels. An increase can be correlated to an
alterationofmembranelipids,proteins,andnucleicacidsandisalso (allreagentsfromInvitrogen,Carlsbad,USA)andmaintainedat37uCand5%CO2.
Cellswereseededatadensityof23104cells/cm2andculturemediumwasreplaced
associated with a wide variety of pathological events including 24hoursafterseedingtoremoveresiduals.TheGFP-taggedvariantofLOX-1was
Figure6|Pro-atherogenicandpro-physiologicaltopographicalenvironments. (a)HUVECsonflatsurfacesadhereinanotpolarizedway.Inthis
conditionLOX-1hasadimericformanddiffusesmoreslowlyinmembrane.Itisactiveinox-LDLbindingandinternalization,disposingHUVECsto
aproatherogeniccondition.(b)HUVECsonnanogratingsadhereinapolarizedway,showingastretchedandalignedmorphology.LOX-1diffuses
fasterandinacontractility-dependentway;furthermoreitisblockedinamonomericformthatisconsideredunabletobindandinternalizeox-LDL,
protectingHUVECsagainstox-LDLinducedatherogenesis.
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transientlytransfectedusingelectroporation(Neon,DigitalBio),accordingto calibratethelaserpowerandscanningconditionsrequiredforthemeasurementof
manufacturer’sinstructions. amonomericproteindiffusingwithinmembranes,wemeasuredthebrightnessof
Formicrotubuleandactin-filamentdisassemblyexperiments,cellsweretreated HUVECstransientlyexpressingthenon-aggregatingfarnesyl-EGFPadduct
with50nMnocodazole(NCZ)and1mMcytochalasinD,respectively,for15min (EGFP-F).TheseconditionswerethenusedfortheLOX-1-GFPexperiments.
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