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Peptides
jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s
Wound healing activity of the human antimicrobial peptide LL37
Reinaldo Ramos
a, João Pedro Silva
a, Ana Cristina Rodrigues
a, Raquel Costa
b, Luísa Guardão
b, Fernando Schmitt
b, Raquel Soares
b, Manuel Vilanova
c,d, Lucília Domingues
a, Miguel Gama
a,∗aIBB,InstituteofBiotechnologyandBioengineering,CentreofBiologicalEngineering,UniversityofMinho,CampusdeGualtar,4710-057Braga,Portugal
bDepartmentofBiochemistry(U38-FCT),FacultyofMedicine,UniversityofPorto,4200-319Porto,Portugal
cICBAS–InstitutodeCiênciasBiomédicasdeAbelSalazar,LargodoProfessorAbelSalazar2,4099-003Porto,Portugal
dIBMC– InstitutodeBiologiaMoleculareCelular,RuadoCampoAlegre823,4150-180Porto,Portugal
a r t i c l e i n f o
Articlehistory:
Received24May2011
Receivedinrevisedform1June2011 Accepted3June2011
Availableonline13June2011
Keywords:
Antimicrobialpeptide LL37
Angiogenesis Woundhealing
a b s t r a c t
Antimicrobialpeptides(AMPs)arepartoftheinnateimmunesystemandaregenerallydefinedascationic, amphipathicpeptides,withlessthan50aminoacids,includingmultiplearginineandlysineresidues.The humancathelicidinantimicrobialpeptideLL37canbefoundatdifferentconcentrationsinmanydifferent cells,tissuesandbodyfluidsandhasabroadspectrumofantimicrobialandimmunomodulatoryactivi- ties.Thehealingofwoundisacomplexprocessthatinvolvesdifferentsteps:hemostasis,inflammation, remodeling/granulationtissueformationandre-epithelialization.Inflammationandangiogenesisare twofundamentalphysiologicalconditionsimplicatedinthisprocess.Wehaverecentlydevelopedanew methodfortheexpressionandpurificationofrecombinantLL37.Inthiswork,weshowthattherecombi- nantpeptideP-LL37withaN-terminusprolinepreservesitsimmunophysiologicalpropertiesinvitroand invivo.P-LL37neutralizedtheactivationofmacrophagesbylipopolysaccharide(LPS).Besides,thepep- tideinducedproliferation,migrationandtubule-likestructuresformationbyendothelialcells.Wound healingexperimentswereperformedindexamethasone-treatedmicetostudytheeffectofLL37onangio- genesisandwoundregeneration.ThetopicalapplicationofsyntheticandrecombinantLL37increased vascularizationandre-epithelialization.Takentogether,theseresultsclearlydemonstratethatLL37may haveakeyroleinwoundregenerationthroughvascularization.
©2011ElsevierInc.Allrightsreserved.
1. Introduction
Cationicantimicrobialpeptides(AMPs)representthefirstline ofdefenseagainstmanyinvadingpathogens.Thesesmallamphi- pathicpeptidesarepartoftheinnateimmunesystemandhavea broad-spectrumactivityagainstbacteria,fungi[1]andviruses[17].
However,directantimicrobialactionisnottheonly,andproba- blynoteventhemostimportanttaskofmammalianAMPs.Infact, thesepeptides presentlowantimicrobialactivitiesunderserum andtissueconditions[16,17].Nevertheless,theyhaveaffinityfor lipopolysaccharide(LPS)andcanpreventfromlethalendotoxemia bysuppressingcytokineproduction[15].Moreover,theseAMPs appeartobeinvolvedintheorchestrationofmanyaspectsofinnate immunityandtheinflammatoryresponse[17].
Inmammals,atleasttwodistinctgroupsofAMPsarefound.
Defensinsarethemorerepresentativesandcathelicidinsformthe secondgroupofmammalianAMPs.Cathelicidinsshare ahighly conservedN-terminalcathelindomain,flankedbyarathervariable
∗Correspondingauthor.Tel.:+351253604418;fax:+351253604429.
E-mailaddress:fmgama@deb.uminho.pt(M.Gama).
antimicrobialpeptideontheC-terminus[32].ThehCAP-18/LL37is theonlyknownhumancathelicidin.Uponstimulation,hCAP-18is cleavedextracellularlybyproteinase3togeneratethepeptideLL37 [36].TheantimicrobialpeptideisreferredtoasLL37,sinceithasa 37aminoacidssequencestartingwithtwoleucines.Itisa4.5kDa, cationic(+6),amphipathic␣-helicalpeptide,withabroadspectrum ofantimicrobialactivity.LL37anditsprecursorhCAP18,werefirst describedinleucocytesandtestis,butweresoonfoundinalarge varietyofcells, tissuesandbody fluids,assummarized byDurr etal.[11].Uponinjuryorinfection,thereisastrongupregulationof hCAP18/LL37,indicatingthatLL37assiststheimmunesystem.The downregulationofLL37hasbeenassociatedwithseveraldiseases:
adeficiencyofLL37intheskinofpatientswithatopicdermatitis [29]orchroniculcers[18]mayaccountfortheincreasedriskfor skininfections.Nizetetal.[28]showedthatmicewithdisrupted Cnlp,thegenecoding forcathelin-relatedantimicrobialpeptide (CRAMP),showedincreasedsusceptibilitytoskininfections.
BesidesitsantimicrobialpropertiesLL37playsacentralrolein innateimmune responsesand inflammation.It hasbeenidenti- fiedasapotentchemoattractantformastcells[27],monocytes, T lymphocytes and neutrophils [42] through formyl peptide receptor-like1(FPRL1).ThefactthatLL37canattractleukocytes contributestohostdefenseagainstinfections.LL37alsopromotes 0196-9781/$–seefrontmatter©2011ElsevierInc.Allrightsreserved.
doi:10.1016/j.peptides.2011.06.005
woundhealing[18],angiogenesisandarteriogenesis[20]andacts asimmuneadjuvant[21].
Acutewounds normallyheal in a veryorderly and efficient mannercharacterizedby four distinct,but overlapping phases:
hemostasis,inflammation,proliferationandremodeling.Allthese phasesarestrictlyconnectedandplayakeyroleforacomplete andproperrestorationoftheinjuredtissue[10].Theinflammatory phaseisinitiatedimmediatelyuponinjury.Severalinflammatory cells migrate to the wound site to fight infection and remove debris.Theproliferativephaseischaracterizedbytheformation ofgranulationtissue,epithelialization,andangiogenesis.Then,the newcollagenmatrixbecomescross-linkedandorganizedduring thefinal remodelingphase. Numerous cell-signalingeventsare requiredtogeneratethisefficientandhighlycontrolledrepairpro- cess.
Inthiswork,wedemonstratethatP-LL37producedbyrecom- binanttechniquesinourlaboratory[33] preservesitsimmuno- physiological properties and promotes wound healing through re-epithelializationandvascularizationindexamethasone-treated mice.
2. Materialsandmethods 2.1. Animals
Animal experiments were conducted according to accepted standardsofhumaneanimalcare(DeclarationofHelsinki,Euro- pean Community guidelines (86/609/EEC) and Portuguese Act (129/92)fortheuseofexperimentalanimals).
2.2. Reagents
All chemicals, media and reagents were purchased from Sigma–Aldrichexcept statedotherwise. Synthetic LL37(LLGDF- FRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was purchased from Bachem, Switzerland. Recombinant P-LL37 (PLLGDFFR- KSKEKIGKEFKRIVQRIKDFLRNLVPRTES)wasexpressedandpurified asdescribedpreviously[33].Briefly,theDNAencodingthefusion protein LK-CBM3-LL37 was successfully cloned in pET21a and expressedinEscherichiacoliBL21DE3(Novagen) inLuria-Broth (LB)medium.Thefusion proteinwaspurifiedoncelluloseCF11 andformicacidwasappliedtocleaveLL37fromthefusioncarrier (LK-CBM3).TheresultingrecombinantP-LL37wasfinallypurified byreverse-phaseHPLC.
2.3. Cultureofmurinebonemarrow-derivedmacrophages (BMDM);suppressionofTNFproductionbyLPS-activated macrophages
Macrophageswereobtainedfrommousebonemarrowasfol- lows:miceweresacrificedandfemursandtibiasremovedunder asepticconditions.BoneswereflushedwithHanks’balancedsalt solution.Theresultingcellsuspensionwascentrifugedat500×g andresuspendedinRPMI1640mediumsupplementedwith10mM HEPES,10%heat-inactivatedfetal bovineserum(FBS), 60g/ml penicillin/streptavidin, 0.005mM -mercaptoethanol (complete RPMI[cRPMI]),and10%L929cellconditionedmedium.Toremove fibroblastsordifferentiatedmacrophages,cellswereculturedon cellculturedishes(Sarstedt,Canada),overnightat37◦Cina5%CO2 atmosphere.Then,nonadherentcellswerecollectedwithwarm cRPMI,centrifugedat500×g,distributedin96-wellplates(Sarst- edt,Canada)atadensityof1×105cells/well,andincubatedat37◦C ina5%CO2atmosphere.Fourdaysafterseeding,10%ofL929cell conditionedmediumwasadded,and themediumwasrenewed onthe seventh day. After 10 days in culture, cells were com- pletelydifferentiatedintomacrophages.Thismethodallowsforthe
differentiationofahomogenousprimarycultureofmacrophages thatretainthemorphological,physiologicalandsurfacemarkers characteristicsofthesephagocyticcells[26,39,43].LPSwasthen incubatedwiththemacrophagesculture.Atthesametime,syn- theticLL37 and P-LL37wereadded at differentconcentrations.
After24h,thesupernatantswereremovedandtestedforTNF-␣ productionbyELISA(eBioscience).
2.4. Cellcultureexperiments
Human microvascularendothelial cells (HMECs) and human umbilicalveinendothelialcells(HUVECs)wereusedintheexperi- ments.HMECswereculturedinRPMI1640mediumsupplemented with10%FBS,1%penicillin/streptomycin,4.76g/lofHEPES,1ml/l ofepidermal growthfactor (EGF) and 1mg/l ofhydrocortisone.
HUVECs were cultured in medium 199 (M199) supplemented with0.1mg/ml of heparin, 20% FBS, 1% penicillin/streptomycin and 2l/ml endothelial cell growth supplement (ECGS). Rat bone-marrowmesenchymalstemcells (MSCs) wereculturedin Dulbecco’smodifiedEagle’smedium(DMEM)supplementedwith 10%FBSand1%penicillin/streptomycin.Thecellslinesweremain- tainedat37◦Cinahumidified5%CO2atmosphere.
2.5. MTStoxicityassay
HMECs (2×105 cells/ml) were grown until 70–90% conflu- ence and incubated with different concentrations of synthetic and recombinant LL37for 24h in 96-well plates. Toxicity was confirmedusing10% DMSO.Cellswerewashed twicewithPBS and their viability was assessed using Cell Titer 96®Aqueous ONE Solution Reagent (MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium]col- orimetric assay (Promega, Madison, USA), according to the instructions provided bythemanufacturer. Opticaldensity was measuredat492nm.Resultsareexpressedaspercentageofcontrol, whichwasconsideredtobe100%.
2.6. BrdUproliferationassay
HMECs and HUVECs (6×104 cells/ml) werecultured in 24- wellplates toadhereovernight.The cells werethen incubated for 48h withsynthetic and recombinant LL37,in thepresence of5-bromodeoxyuridine(BrdU)solutionatafinalconcentration of0.01mM.ProliferationwasquantifiedusingIn-SituDetection Kit(BDBiosciencesPharmigen,USA),accordingtomanufacturer’s instructions.Theresultsaregivenasmean±SEMandareexpressed aspercentageofcontrol,whichwasconsideredtobe100%.
2.7. Migrationanalysis
Migration assays were performed in transwell BD-matrigel basement membrane matrix inserts (BD-Biosciences, Belgium).
ThechemotacticcapacityofLL37wasevaluatedbycountingthe cells that migrated through the membranes. Transwell inserts containingan8mpore-sizePETmembranecoatedwithauni- formlayerofmatrigelbasementmembrane wereused.HUVECs (5×104cells/ml)wereseededoninsertsinserum-freemedium, and placed on wells containing medium complemented with FBS2%andrecombinantorsyntheticLL37ata concentrationof 5g/ml.For MSCs (5×104cells/ml),theinsertswere placed on wellscontainingDMEMwithBSA0.1%andrecombinantLL37at aconcentrationof5g/ml.Afterincubationfor24hmembranes wereremovedfrominserts,stainedwithDAPI–methanolfor5min andvisualizedunderafluorescencemicroscope.Theinvadingcells
ofeachmembranewerecountedandtheresultsareexpressedas percentageofcontrol,whichwasconsideredtobe100%.
2.8. Angiogenesis
TheabilityofLL37topromotetheformationofcapillary-like structureswasexaminedinMatrigelassay.HMECsandHUVECs (3×104 cells/well)wereincubatedwith5g/mlofrecombinant andsyntheticLL37for24honmatrigel-coated96-wellplates.The capillary-likestructuresineachwellwerethencountedandresults areexpressedaspercentageofcontrol,whichwasconsideredtobe 100%.
2.9. Woundhealingassay
C57BL/6mice (purchasedatCharles River,Wilmington, MA) with8–12weeksoldwereusedinthisstudy.Aftergeneralanes- thesia,haironthedorsalsideoftheanimalswasshavedandthe skinwascleanedwith70%ethanol.A5mmskinbiopsypunchwas usedtocreatefullthicknesscutaneouswoundsunderasepticcon- ditions.Twowoundswerecreatedonthedorsalsurfacewithone oneachsideofthemidline.LL37(recombinantorsynthetic)and sterilewaterwereappliedtopicallytwiceaday(2×10gin50l).
Woundswereleftopenandnotcoveredbyanytypeofdressing.
Toimpairhealing,miceweretreateddailywithdexamethasone (0.25mg/kgbodyweight,intramuscularly).Micewereexamined daily for wound-healing progression. After 7 days, theanimals wereeuthanizedandwoundtissuewascollectedforhistological andimmunohistochemistrystudies.Skinwoundtissuespecimens werefixedin10%neutral-bufferedformalinsolutionandparaffin- embedded.Histologicalanalyzeswereperformedin5-mtissue sections.
2.10. Immunohistochemistry
Microvesseldensity(MVD)wasevaluatedineachformalin-fixed paraffin-embeddedwoundedtissuesectionbyimmunohistochem- istry.Tissueslideswereincubatedovernight8hwithananti-CD31 antibody.Briefly,sixareaswiththehighestvisiblebloodvesselden- sity(markedbythevesselmarker)persectionwereselected,and thenumberofbloodvesselswascountedperhigh-powerfield.
3. Results
3.1. RecombinantP-LL37neutralizestheactivationof macrophagesbyLPS
Murinemacrophagesareknowntoinduceseveralcytokines, includingtumornecrosisfactor-␣(TNF-␣),uponLPSactivation.LPS wasaddedtomacrophageculturestoaconcentrationof10ng/ml, either alone or in the presence of 20g/ml of recombinant P- LL37orsyntheticLL37.Cultureswerethenincubatedfor24h.The absenceofLPSintherecombinantproteinwasconfirmedusingthe limulusamoebocytelysatetest(datanotshown).Asexpected,the recombinantP-LL37,aswellasthesyntheticpeptide,neutralized theLPS-mediatedactivationofmacrophages(Fig.1).
3.2. Toxicity
In ordertoevaluatethe cytotoxicityof recombinantP-LL37, HMECswereculturedfor24hinthepresenceofthepeptideandthe cellviabilitywasevaluatedbyMTS.DMSO10%wasusedascyto- toxicagent,apositivecontrol.AsshowninFig.2,P-LL37didnot affectviabilityofHMECsatanyoftheconcentrationsused.Actually, with50and500ng/mlofP-LL37,aslightincreaseofviabilitywas observed,indicatingthattherecombinantpeptideP-LL37might
Fig.1. RecombinantandsyntheticLL37inhibitstheTNF-␣productionbyLPS- activatedmacrophages.***P≤0.001vs.control.
havepromotedproliferation,aspreviouslyshownbyKoczullaetal.
[20].
3.3. P-LL37promotesproliferation,migrationandtubule-like structuresformationbyendothelialcells
ProliferationofHMECsandHUVECswasevaluatedusingthe BrdUincorporationassay.SyntheticLL37(5g/ml)wasusedasa positivecontroland100Mofdeoxycholicacid(DCA),anantag- onistofFPRL-1,asnegativecontrol.DCAattenuatestheactivation ofFPRL-1,bybindingtothecellmembraneandinterferingwith theaccessofthereceptortoitsagonists[23].Fig.3demonstrates thatP-LL37promotedproliferationwithconcentrationsaslowas 50ng/mlinHMECs(a)andtheeffectat5g/mlwasverysimilar totheoneobtainedusingthesyntheticLL37.Theproliferationof HUVECs(b)wasinferiorbutonceagaintherewerenosignificant differencesbetweensyntheticandrecombinantpeptides.
Next,thechemotacticandangiogenicactivitiesofP-LL37were evaluated.Fig. 4illustrates theinvasion assays performedwith HUVECs(a)andMSCs(b).Again,P-LL37wasabletoincreasecell invasivenesscapacitybothinHUVECsandinMSCs.Thecapacityof LL37topromotetheformationofcapillarystructureswasassessed withHUVECsandHMECsusingmatrigelasthree-dimensionalscaf- fold(Fig.5).TheuseofLL37hadastrongeffectinangiogenesis.In fact,italmostdoubledthenumberofcapillariesformedbyHUVECs andHMECs.
3.4. Woundhealingassays
To further examine the effects and biological activities of syntheticandrecombinantLL37,woundhealingexperimentsin dexamethasone-treated mice were performed. The peptides or vehicleweretopicallyapplieddailyfor7daysinthe5mmskin wounds.Dexamethasonewasusedtoimpairhealing.Fig.6illus-
Fig.2. CytotoxicityofP-LL37inconfluentHMECsusingMTSassay.Threeindepen- dentexperimentswereperformedinduplicate.***P≤0.001vs.control.
Fig.3. EffectsofsyntheticandrecombinantLL37inHMECs(a)andHUVECs(b)proliferationassessedbyBrdUassay.P-LL37andsyntheticLL37promotedsimilarproliferation ofendothelialcells.Threeindependentexperimentswereperformedinduplicate.*P≤0.05and**P≤0.01vs.control.
Fig.4.InvadingcellsrelativetotheinitialamountofHUVECs(a)andMSCs(b)culturedusingadouble-chamberassay.Resultsaremeans±SEMofthreeindependent experimentsperformedinduplicateandexpressedrelativelytocontrol.*P≤0.05and**P≤0.01vs.control.
tratesthewoundsafterdailytreatmentwithrecombinantLL37 (twoapplicationsof10geach)orvehicle,ondayseven.Theeffect ofthepeptideonwoundclosureisverystrongwhencomparingto control.
Thewoundsectionsstainedwithhematoxylin andeosin are showninFig.7.Nosignificantdifferencesarefoundinthegran- ulationtissuewithahighnumberofgranulocytesinbothcases.
However,regardingre-epithelialization,thelesionstreatedwith
controlor syntheticandrecombinant LL37presentlargediffer- ences. In the control, it is clear that the keranocytes layer is incomplete.On thecontrary,in thewounds treatedwithLL37, there-epithelializationisalmostcomplete,demonstratingthatthe healingprocessisaccelerated.More,ahighnumberofbloodves- selscanbeidentifiedinthewoundstreatedwithLL37(pointed by the arrows), indicating that synthetic or recombinant LL37 strongly promote angiogenesisin vivo.These resultswere ver-
Fig.5.RecombinantandsyntheticLL37promoteangiogenesisinvitro.(Left)Capillary-likestructuresformationofHMECs,aftertreatmentwith5g/mlofLL37,visualized underaphase-contrastmicroscope.Figuresarerepresentativeofthewholecultures.(Right)Quantificationofcapillary-likestructuresassembly.Resultsaremeans±SEMof threeindependentexperimentsperformedintriplicateandexpressedrelativelytocontrol.*P≤0.05and**P≤0.01vs.control.
Fig.6.Macroscopicexaminationofthewoundsatdayseven.
ified byimmunohistochemistry(Fig.8).The angiogenicprocess was confirmed,in ourexperimental model, by theaugmented microvasculardensityevaluatedbythemeansofnewlyformed vesselscountedafterimmunostaining.Thewoundstreatedwith eithersyntheticorrecombinantLL37possessasignificantlyhigher numberofnewbloodvessels.Moreover,thevesselsinthetissues treatedwiththepeptidearemuchlarger.Severalexperimentswere performedwithbothrecombinantandsyntheticLL37andsimilar resultswereobserved.Theseresultsclearlydemonstratetheability ofLL37topromotewoundhealingasaverypromisingresult.
4. Discussion
A variety of endotoxins released by the outer mem- braneofGram-negativebacteria,namelythelipopolysaccharide (LPS)–proteincomplex,playanimportantroleinpathogenesisof manyexogenousrespiratorydiseases.ThemassivereleaseofLPS canleadinextremecasestoendotoxicshockandtherefore,death [14].Manyantibioticsreleaseendotoxinbydisruptingthecellwall [9,31],andpatientscuredofbacterialinfectionscansufferfrom endotoxicshock.Theactivationofmacrophagesisstartedwhen LPSbinds theLPS-bindingprotein(LBP). Inturn, LBPbindsthe macrophagesurfaceprotein CD14,theprimaryreceptor ofLPS, andis100-foldmorepotentthanLPSalone[34].Theantimicro- bialactivityofthepeptideLL37resultsfromthedisruptionofthe bacteriacellwall;however,LL37canalsoneutralizeLPSactivityby forminghighaffinitycomplexes[22,35].
TheTNF-␣productionwasverylowinthepresenceofeither therecombinantorthesyntheticpeptides,indicatingthatrecom- binantP-LL37hasaffinityforLPS.Therecombinantpeptideonly differsfrom thesynthetic in one amino acid, a neutral proline resulting from the formic acid hydrolysis [33]. Therefore, the globalnetcharge(+5)remainsunaltered.Sinceelectrostaticand hydrophobicinteractionsarethepredominantforcesdrivingthe bindingbetweenantimicrobialpeptidesandLPS,theseresultswere expected.However,Rosenfeldetal.[34]describedthata strong bindingof AMPs–LPSmight notbesufficient toneutralizeLPS- inducedmacrophageactivation.Infact,thoseauthorsshowedthat
theAMPmagainin,althoughbindingLPSwithhighaffinity,didnot reducetheproductionofTNF-␣.SomeAMPs,includingLL37,can competewithLPSonitsbindingsite withintheCD14receptor, modulatingtheTLRsignalingpathwayinvolvingNFB[25].There- fore,theinhibitionofTNF-␣productionbyLL37isnotonlycaused bytheinteractionwithLPS.Overall,theseresultsdemonstratethat therecombinantpeptideP-LL37isbiologicallyactive.
ThehumanantimicrobialpeptideLL37wasinitiallyrecognized foritsantimicrobialproperties.Infact,ithasabroadspectrumof antimicrobialactivityagainstbacteria,fungi,andviralpathogens, assummarizedbyDurretal.[11].Severalstudiesreportedthat AMPs loosetheirantimicrobialactivityinelevatedsalt concen- trationsand underserumconditions[2,13,24].Thisinhibitionis lesspronouncedinthecaseofLL37,probablyduetoits␣-helical structure[30];however,thepeptidebecomesinactiveagainstcer- tainorganisms–susceptibleunderlowsaltconditions–inmedia with100mMNaCl[38].Furthermore,LL37hadnokillingactiv- ityagainstStaphylococcusaureusorSalmonellatyphimuriuminthe presenceoftissue-culturemedium[5].Therefore,therelevanceof antimicrobialactivityinvivoisnotclear.However,AMPsareimper- ativeininnateimmunity,sinceanimalslackinggeneexpressionof thesepeptidesweremoresusceptibletoinfections[28].Besidesits antimicrobialproperties,manyotheractivitieshavebeenreported forthehumanpeptideLL37.Asalreadystated,LL37isinvolvedin manyaspectsofinnateimmunity,likechemotaxis,angiogenesisor woundhealing.Althoughnotcompletelyunderstood,theseactivi- tieshavebeenassociatedtosomecellsreceptorslikeFPRL-1,P2X7
andepidermalgrowthfactorreceptor(EGFR)[12,19,37].Among these,LL37onlybindsdirectlytoFPRL-1[42],inducingcellularsig- nalingandCa2+flux[20].So,wehavetestedtheeffectsofP-LL37 in proliferation,migration and angiogenesisof endothelialcells toascertainwhethertherecombinantpeptidebindsthereceptor FPRL-1.TheresultsconfirmedthatP-LL37promotesproliferation, migrationandtubule-likestructuresformationbyendothelialcells.
TheeffectofP-LL37inthemigrationofHUVECsandMSCsis significant,withanincreaseofalmost100%invadingcellsbeing observedinbothcases.IntheexperimentswithHUVECs,synthetic LL37promotedaverystrongincreaseininvadingcapacity,much superiorthantheoneobtainedwithP-LL37.Thisresultwassurpris- ing,sincenosignificantdifferenceswerefoundintheproliferation assays.P-LL37isobtainedbyrecombinanttechniquesandthebatch usedinthis experimentcouldnotbeaspureasexpected,mis- leadingtheconcentrationquantification.Asmallerconcentration oftherecombinantpeptidemighthavebeenused,whichwould explainthis result.Infact,asillustratedinFig.5,theformation ofcapillarystructureswasverysimilarusingbothpeptides.The invasionassaywithMSCswasonlyperformedwithcontroland recombinantLL37.Coffeltetal.[8]reportedthatLL37wasableto chemoattractMSCsthroughtheactivationofFPRL-1.Considering alltheresultsobtained,weclearlydemonstratethattherecombi- nantLL37producedinourlaboratoryisfullyactiveinvitro.The N-terminusproline,resultoftheformicacidhydrolysis,doesnot interferewiththebindingofLL37–LPSandtothereceptorFPRL-1.
Glucocorticoids affect almostevery phase of wound healing becauseoftheirinhibitoryeffectongene expressioninvarious cells [40]. Beer et al. [3] showed that dexamethasone inhibits recruitmentofvariousinflammatorycellsandgeneexpressionof manykeywoundhealingcytokinesandgrowthfactors.Duringthe proliferativeandremodelingphases,dexamethasonecaninhibit thesynthesis ofseveraldermalextracellular matrix(ECM) pro- teins,and delayre-epithelializationand fibroplasia.Thehealing ofaskinwoundisacomplexprocessrequiringthecollaborative efforts of many differenttissues and celllineages and involves the formationof new extracellular matrix, cell infiltration,and tissue remodeling.Inflammationand angiogenesisaretwo fun- damentalphysiological conditionsimplicated inthis process.In
Fig.7. Hematoxylinandeosinstainedmicrographsofwoundtissuesectionstreatedwithcontrol,syntheticandrecombinantLL37.Therectangleidentifiesanareafurther investigatedinahighermagnificationpicture.Atdayseven,re-epithelializationisalmostcompleteinwoundtreatedwithLL37andahighnumberofbloodvesselsare present(pointedbyarrows).
fact,theformationofnewbloodvesselsisaprerequisiteoftis- suerepairandwoundhealing[6].Asalreadystatedandconfirmed inthiswork,LL37promotesangiogenesis.Moreover,LL37appears tohaveaveryimportantroleinwoundhealing.Infact,Heilborn et al. [18] showed that the useof LL37antibody inhibited re- epithelializationinaconcentration-dependentmannerinanorgan culturedfull-thicknessexvivowoundmodel.Moreover,Carretero etal.[7]transferredanadenoviralvectorcontainingLL37viaintra- dermalinjectiontoexcisionalwoundsinob/obmice,improving re-epithelializationandgranulationtissueformation.Considering alltheseresults, we decidedto studytheeffects of thetopical applicationofLL37onwounds.SyntheticandrecombinantLL37 stronglyacceleratedthehealingprocesswithare-epithelialization almostcompleteafter7days.Moreover,asignificantnumberof newbloodvesselswerepresentinthewoundstreatedwiththe peptide.
TheimportanceofLL37inwoundhealinghasalreadybeenpre- sented,but thetopicalapplicationof LL37onskinlesionswith impairedhealing hasnot beenstudiedsofar.In this work,we demonstratethatLL37canbeusedtopicallytosupportwoundheal- ing.Thetopicalapplicationofthepeptideallowsabetterregulation oftheprocess.Theappliedconcentrationscanbeeasilycontrolled andtheadministrationcanbestoppedatanymoment.Moreover, deliverysystemscanbeused toobtaina regulatedadministra- tion.ThetightregulationofAMPsisveryimportantsinceelevated amountsmaylead toachronic inflammatoryprocess, asit has beendemonstratedforpsoriasisandrosacea[29,41].Inorderto betterunderstandtheroleofLL37inwoundhealing,furtherexper- imentswillbeperformed,namelywithdiabeticmice.Diabeticmice haveimpairedhealinganditiswellknownthatthehealingtreat- mentsofdiabeticulcersdidnotdeliversatisfactoryresults.Inthis typeoflesions,angiogenesisseemstobethemostcompromised
Fig.8. (Left)RepresentativeimmunostainingofskinwoundssamplestreatedwithvehicleorLL37atdayseven.Arrowsindicatethebloodvessels.(Right)Quantificationof bloodvessels.Resultsaremeans±SEMofthreeindependentexperimentsandareexpressedaspercentageofcontrol.***P≤0.001vs.control.
phasewithimpairmentinVEGFsynthesis[4].Regardingtheposi- tiveresultsobtainedinthiswork,webelievethatthetreatmentof diabeticulcerswithLL37willimprovewoundhealing.
Conflictofinterest
Allauthorsdisclosethatthereisnoconflictofinterestinclud- inganyfinancial,personalorotherrelationshipswithotherpeople ororganizations.Allauthorshavemateriallyparticipatedinthe researchand/orarticlepreparation.Allauthorshaveapprovedthe finalarticle.
Acknowledgements
Thiswork wassupported by theindividual Grant SFRH/BD/
27404/2006fromFundac¸ãoparaaCiênciaeaTecnologia(Portugal).
WearegratefultoJoanaAlmeida,attheDepartmentofBiochem- istry,UniversityofPorto,forthehelpwiththehistologicalanalyzes.
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