www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Sulfated
glycosaminoglycans
in
human
vocal
fold
lamina
propria
夽
Sung
Woo
Park
a,
Gustavo
Polacow
Korn
a,
Elsa
Yoko
Kobayashi
b,
João
Roberto
Maciel
Martins
b,c,∗,
Noemi
Grigoletto
De
Biase
a,d,∗aUniversidadeFederaldeSãoPaulo(UNIFESP/EPM),EscolaPaulistadeMedicina,DepartamentodeOtorrinolaringologiae
CirurgiadeCabec¸aePescoc¸o,SetordeLaringeeVoz,SãoPaulo,SP,Brazil
bUniversidadeFederaldeSãoPaulo(UNIFESP/EPM),EscolaPaulistadeMedicina,DepartamentodeBioquímica,
DivisãodeBiologiaMolecular,SãoPaulo,SP,Brazil
cUniversidadeFederaldeSãoPaulo(UNIFESP/EPM),EscolaPaulistadeMedicina,DepartamentodeMedicina,
DivisãodeEndocrinologiaeMetabologia,SãoPaulo,SP,Brazil
dPontifíciaUniversidadeCatólicadeSãoPaulo(PUC-SP),SãoPaulo,SP,Brazil
Received5May2016;accepted10May2016 Availableonline2June2016
KEYWORDS
Larynx;
Glycosaminoglycans; Vocalfolds
Abstract
Introduction:Thedistribution,concentrationandfunctionofglycosaminoglycansinthevarious vocalfoldtissuesarestillunclear.
Objective:Toevaluate thedistributionandconcentrationofsulfated glycosaminoglycansin differentlayersofthehumanvocalfoldaccordingtogenderandage.
Methods:Weused11vocalfoldsobtainedfromcadavers(7menand4women)withnolaryngeal lesion,lessthan12hafter death,andagedbetween35 and98years.Thefolds underwent glycosaminoglycansextractionfromthecoverandligament,andpost-electrophoresisanalysis. Datawerecomparedaccordingtothelayer,ageandgender.
Results:Theconcentrationofdermatansulfatewassignificantlyhigherinalllayers.No dif-ferenceswere observed inthetotal concentrations ofglycosaminoglycansinlayersstudied accordingtogender.Itissignificantlylowerinthecoverofindividualsagedbelow60years.
Conclusion:Dermatansulfate,chondroitinsulfate,andheparansulfatewereobservedinthe humanvocalfoldscover andligamentofbothgenders,with theconcentration ofdermatan sulfatebeingsignificantlyhigherinalllayers.Glycosaminoglycansconcentrationonthecover issignificantlylowerinindividualsbelow60yearscomparedwithelderly.
© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).
夽 Pleasecitethisarticleas:ParkSW,KornGP,KobayashiEY,MartinsJR,DeBiaseNG.Sulfatedglycosaminoglycansinhumanvocalfold
laminapropria.BrazJOtorhinolaryngol.2017;83:426---31.
∗Correspondingauthors.
E-mails:jrmacmartins@yahoo.com.br(J.R.Martins),ngdebiase@gmail.com(N.G.DeBiase).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCervico-Facial.
http://dx.doi.org/10.1016/j.bjorl.2016.05.003
PALAVRAS-CHAVE
Laringe;
Glicosaminoglicanas; Pregasvocais
Glicosaminoglicanossulfatadosnalâminaprópriadepregavocalhumana
Resumo
Introduc¸ão: Adistribuic¸ão,concentrac¸ãoefunc¸ãodosglicosaminoglicanosnosdiversostecidos dapregavocalaindanãoestáesclarecida.
Objetivo: Avaliaradistribuic¸ãoeconcentrac¸ãodosglicosaminoglicanossulfatadosnas difer-entescamadasdapregavocalhumanadeacordocomosexoeaidade.
Método: Foramutilizadas11pregasvocaisobtidasdecadáveres(setehomensequatro mu-lheres)semlesãodelaringe,commenosde12horasdeóbitoecomidadeentre35e98anos. As pregasforam submetidas àextrac¸ãode glicosaminoglicanos dacobertura eligamentoe leiturapóseletroforese.Osdadosforamcomparadossegundoacamada,idadeesexo.
Resultados: A concentrac¸ão de Dermatan sulfato foi significativamente maior em todas as camadas.Nãoforamobservadasdiferenc¸as nasconcentrac¸õestotais deglicosaminoglicanos nascamadasestudadasquantoaogênero.Ésignificantementemenoremindivíduoscomidade abaixode60anosnacobertura.
Conclusão:Dermatam sulfato, condroitim sulfato e heparam sulfato foram observados na coberturaeligamentodepregasvocaishumanas, deambosossexos,sendoaconcentrac¸ão dedermatam sulfatosignificativamentemaiorem todasascamadas.Aconcentrac¸ãode gli-cosaminoglicanosnacoberturaésignificativamentemenoremindivíduosabaixode60anosem comparac¸ãocomidosos.
© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).
Introduction
Thevocalfoldsareadaptedforspeechfunction.The vibra-tionofthevocalfoldsdependsonitshistologicalstructure, especiallyofthelaminapropria(LP),stratifiedinthefree edge regionin threelayers: superficial,intermediate and deep.Allofthemconsistofcellsandextracellularmatrix, differingqualitativelyand quantitativelyfromeach other, especially astothe fibrous proteins (collagen and elastic fibers)andinterstitialproteins.1
Among theinterstitialproteins, theglycosaminoglycans (GAGs), proteoglycans (PG), and non-collagenous glyco-proteinsarenotable.Theyinfluenceviscosity,hydrationand tissuevolume.2,3 The differencesinthearrangementsand
quantities ofcomponentsof theextracellular matrix, and also the different interactions of these components with each otherandthe cells,aredynamicallyadjusted tothe functional demands of each tissue.4---6 In the case of the
vocalfolds,thisfunctionaldemandistheproductionofthe mucosalwaveand,consequently,thevibrationofthetissue andtheformationofthesoundwave.7
Pawlaketal.2werethefirsttostudythePGsandGAGs
in vocal foldLP. The expression of hyaluronicacid recep-tor,keratansulfate,chondroitinsulfate,heparansulfatePG, anddecorinwasobservedinvariousregions,aswellasthe celltypesintheLP.
Paulsenetal.8observedareductionofsulfatedGAGsin
thetendons(extremities)ofthevocalligament.Agreater lossofGAGinthevocalligamenttendonwasobservedwith aging.Thesestructuresthuslosetheirabilitytoretainwater andtheviscoelasticityofthevocalfoldisimpaired. Ham-mond etal.,9 usedan indirect method tocompare tissue
thatisincubatedor notincubatedwithhyaluronidase and
observedthattheconcentrationofhyaluronicacid(HA)in theLP washigherinmalescomparedtofemales.The dis-tribution also varied according to the layers and gender, beingmoreevenlydistributedamongmalesandmore con-centratedinthedeeperlayersinfemales.10 Usingadirect
method,Lebletal.11andKornetal.12foundahigher
concen-trationofHAinfemaleLPinrelationtomaleandatrendfor adecreasewithaging.Sincebovinetesticularhyaluronidase isnotspecificforHA,itmayhavebrokenotherGAGspresent intheLP,suchaschondroitinsulfate,whichwouldexplain thedifferencefound inthetwostudiesiftherewerealso differencesin theconcentrations of the other interstitial proteins.
Knowledgeofthedistributionandconcentrationof inter-stitialmacromoleculesthathave differentcharacteristics, featuresandeffectsonthefunctionallystratifiedLPlayers, willhelptounderstandthenormalphysiologyofphonation, agingandin pathologicalprocessesthathavean impaired mucosalwave.
Thus,theobjectiveofthisstudyistoinvestigatethe dis-tributionandconcentrationofsulfatedGAGsinthedifferent layersofthevocalfoldaccordingtogenderandage.
Methods
Prospectiveexperimentalstudy
refrigerationtothelaboratorywheretheyweredissected. The pieceswere evaluatedwiththe use of acameraand videosystemwitha25×magnification,andthoseinwhich anyinjuriesorstructuralchanges,suchaspolyps,furrows, leukoplakiawereobservedwereexcludedfromthisstudy.
Avocal foldfromeach individualwasusedat random. Thevocalfoldsunderwentdecorticationwiththehelpofthe videosystem.Threefragmentswereisolated:cover (epithe-liumandsuperficiallayeroftheLP),ligament(intermediate anddeeplayersoftheLP)andmuscle.Fromthese,thecover andligamentwereused.
FortheGAGextractionfromtissues,thefragmentofthe givenlayerofthevocalfoldwasperforatedwithascalpel, homogenized,anddehydratedinacetonefor18hatroom temperature.Then, excess acetone wasremoved by cen-trifugation,4000×g, 15min, and theprecipitate dried in anoven.Foreach1mgofdrypowder,100Lofmaxatase,
4mg/mLin0.05MTris---HCl+1MNaClwereadded,pH8,and incubatedat60◦Cfor18h.Afterincubation,proteasewas
inactivatedby heating at 100◦C, 20min. To the solution,
90%trichloroaceticacidwasaddedundercoolingtoafinal concentrationof 10%. The solution washomogenized and leftat4◦
Cfor10min.Afterthisperiod,itwascentrifugated at4000×gfor15min.Thesupernatantwasseparatedand addedtoabsoluteethanol(3volumes),andthefinal solu-tionmaintainedat−20◦Cforatleast3h.Afterthis,itwas
centrifugatedat 10,000×g,30min,andtheobtained pre-cipitatewasdriedundervacuumandstoredat−20◦Cuntil
analysis.13,14
Then, the precipitate obtained in the previous proce-dureswasresuspended againin distilledwater (10Lper
1mgofdrytissue),and5Lofthissolutionwereappliedin
agarosegel blades,1,3-diaminopropanoacetatebuffer, pH 9,andunderwentelectrophoresis(5V/cm,1h,under refrig-eration) for separation of various GAGs.10,11 As standard,
a mixture of GAGs 1g/mL (heparan sulfate+dermatan
sulfate+chondroitin sulfate) was used. Following elec-trophoresis, the gel was dried under ventilation at room temperature, and stained with 0.1% toluidine blue in 1% acetic acid and 50% ethanol. After 10min, the gel was destained with acetic acid solution 1% and 50% ethanol; driedatroomtemperature,andtheobtainedbands, quanti-fiedbydensitometry(Fig.1).TheconcentrationofGAGwas expresseding/gofdrytissue.15
Datawasstatistically analyzed.To compare the values of GAG concentration according to gender and age the nonparametric Mann---Whitney test was used.To compare the values of GAG concentration in the different layers of the vocal fold the Wilcoxon nonparametric test was used.Descriptive statistics of quantitativevariables were performed: concentration bylayer through the meanand median;andstandarddeviation,minimumandmaximumto showvariability.Also,theAnalysisofVariancewithRepeated Measures(univariate)wasusedasthe observationsinthe differentlayersarerelatedtothesameindividual.To com-pare the concentration of GAG among the layers of the vocal fold, the covariance matrix of the observations of thesameindividualshouldhaveaspecificform.Whenthis particular form wasnot found, and becauseof the small samplesize,thetestwascorrectedbasedonHuynh---Feldt correction.Comparisonsamongconcentrationsweremade using the Bonferroni test. The existence of interaction
CS -
DS -
HS -
Figure1 Bandsobtainedfollowing0.1%toluidineblue stain-ing in a solution of 1% acetic acid, and 50% ethanol and destaining with 1% acetic acidand50% ethanol, which were quantifiedbydensitometry.Thestandardisontheleftofthe image(arrow).CS,condroitinsulfate;DS,dermatansulfate;HS, heparansulfate.
(interaction effect)in the results was alsochecked, i.e., different behaviorsaccording toanother variable.For all tests,asignificancelevelof5%(p≤0.05)wasconsidered.
Results
ThepresentstudyevaluatedVocalFolds(vocalfolds)of11 subjects,4femaleand7male.Fivesubjectswere60years ofageoryoungerandtheother6individualswereover60 yearsofage.
Therewasnosignificant differencebetween the cover andtheligamentwithrespecttotheconcentrationof sul-fatedGAGs(Table1).
Therewasnosignificantdifferenceintheconcentration ofsulfatedGAGsinthelayersofcoverandligament accord-ingtogender(Table2).
ComparingtheconcentrationofsulfatedGAGsastoage range,inthedifferentlayers,therewassignificant differ-enceinthecover.Patientsover60yearshavesignificantly
Table 1 GAGconcentration inthe vocal fold cover and ligament.
Variables GAGs(g/gdryweight)
Cover
Mean±standarddeviation 302.7±224.9 Median(Minimum−Maximum) 217.3(83.9---802.9)
Total 11
Ligament
Mean±standarddeviation 379.3±198.6 Median(minimum−maximum) 322.5(117---681)
Total 11
pvaluea 0.334
a pvalue of nonparametric paired-sample Wilcoxon
Table2 GAGconcentrationaccordingtogenderinthecoverandligament.
Variablespergender Female
GAGs(g/gdryweight)
Male
GAGs(g/gdryweight)
p-valuea
Cover
Mean±standarddeviation 346.8±307 277.5±187.4 0.571
Median(minimum−maximum) 224.4(135.7---802.9) 182.2(83.9---535.2)
Total 4 7
Ligament
Mean±Standarddeviation 490.1±222.3 316±167.9 0.257
Median(minimum−maximum) 554.9(169.7---681) 280.6(117---632.1)
Total 4 7
a p-valueofnonparametricMann---Whitneytest.
Table3 GAGconcentrationaccordingtotheagerangein:coverandligament.
Variablesperagerange Over60years Upto60years p-value
Cover
Mean±standarddeviation 436.6±226.6 141.9±54.7 0.018a
Median(minimum−maximum) 441.1(182.2---802.9) 135.7(83.9---231.4)
Total 6 5
Ligament
Mean±standarddeviation 410.3±201.2 342.1±211.9 0.584 Median(minimum−maximum) 429.2(117---632.1) 236.1(169.7---681)
Total 6 5
a p-valueofthenonparametricMann---Whitneytest.
highervaluesthanthose60yearsandyounger(Table3and
Fig.2).
When comparingtheconcentrationofCS,DSandHSin thecoverandligament,therewasahigherconcentrationof DSinthedifferentlayers,butwithnosignificantdifference inthecomparisonbetweenthelayers(Tables4and5).
GAGs (µg/g dry weight)
Up to 60 years
Age range 250.0
500.0 750.0 1000.0
Surface layer
Over 60 years
∗
Figure2 Box-plotofcoveraccordingtoagerange.
Table5referstotheGAGconcentrationaccordingtothe ageandgenderinthelayersofcoverandligament. Statis-ticalanalysiswasnotpossibleduetothenumberof vocal foldsineachcategory.
Discussion
Somecomponentsofthe extracellularmatrix inthe vocal foldsarebetterknownandstudied,suchaselastinand colla-gen,andmorerecentlytheHA.However,othercomponents oftheextracellularmatrix,suchasGAGsandPGs,havenot beenstudiedfortheirroleandimportanceinthephysiology ofthehumanvocalfolds.
Thus, considering the physiology of vocal fold in the productionof voiceandthe distinctivepropertiesalready known,thisstudyexaminedthepresenceandconcentration ofsulfatedGAGsinhumanvocalfold:onthecover,inwhich thesuperficial layerof LP predominates, andonthe liga-ment.The coverand theligamenthave distinct functions in the mucosal wave formation, and a typical histologi-calconstitution comparedtofibrousproteins, elastin and collagen.1,7Theseparationbetweencoverageandligament,
andbetweenligamentandmuscle,is easilyperformed by dissectionwithmagnification.
Table4 GAGconcentration(CS,DSeHS)inthedifferentlayersofthevocalfold.
Variablesperconcentration CS DS HS p-valuea
Cover
Mean±standarddeviation 29.9±28.8 233.7±187.8 39.1±36.7 <0.001 Median(minimum−maximum) 19.1(0---82.5) 178.9(70.2---670.1) 27.2(0---110.5)
Total 11 11 11
Multiplecomparisons
CS×DS 0.009
CS×HS 1.000
DS×HS 0.009
Ligament
Mean±standarddeviation 45.6±54.4 273.8±119 60±55.3 <0.001 Median(minimum−maximum) 23.9(2.9---156) 289.2(103.5---444.3) 48.5(5.7---161.1)
Total 11 11 11
Multiplecomparisons
CS×DS 0.009
CS×HS 1.000
DS×HS 0.009
p-valueb 0.477 0.374 0.657
ap-valueofnonparametricsample-pairedWilcoxonsigned-ranktest.
b Multiplecomparisons:pvalueofnonparametricsample-pairedWilcoxonsigned-ranktest.
Table5 GAGconcentrationaccordingtogenderandagerangein:coverandligament.
Females Males
Upto60years Over60years Upto60years Over60years
Cover
Mean±standarddeviation 183.6±67.7 510.1±414.1 114.2±28.5 399.9±151.9 Median(minimum−maximum) 183.6(135.7---231.4) 510.1(217.3---802.9) 118.2(83.9---140.5) 441.1(182.2---535.2)
Total 2 2 3 4
Ligament
Mean±standarddeviation 425.4±361.5 554.9±26.7 286.6±113.4 338±215.2 Median(minimum−maximum) 425.4(169.7---681) 554.9(536---573.8) 236.1(207.1---416.4) 301.5(117---632.1)
Total 2 2 3 4
also found no difference in the total GAG in relation to gender.16,17
However,considering theage,a statisticallysignificant differencebetweentheconcentrationsofGAGsinthecover wasseen, withhigher values in individuals over 60 years (Table3).Thehighest concentrationofGAGintheelderly may alsobe related to the decrease of other substances in the vocal folds, such asthe HA itself, as observed by Lebl et al.11 Korn et al.,12 because its concentration is
calculated in terms of g/dry weight of the vocal fold
material.Theligamentshowsnostatisticallysignificant dif-ferencein the concentrationof GAG,by either gender or age(Tables2and3).Paulsen etal.8observedareduction
ofsulfatedGAGsinthevocalligamenttendons,thatis,on theendsoftheligament,aswellasmorepronouncedloss ofGAGintendonvocalligamentwiththeagingprocess.
Hammondetal.9in astudythatlookedat the
concen-trationofelastinfoundinvocalfoldswithageandgender, found no significant difference in terms of gender, but
an important and significant difference as for the age of patients. Adult vocal folds showed less elastin than the elderly. However,thisincrease inelastin density does not necessarilymeananincreaseinvocalfoldselasticity,since thefibersobservedintheelderlyhadachangedstructure.
Thus,changeoftheelastinstructure,theconcentration of HA and sulfated GAGs may be related to tissue aging withconsequentrepercussionsontheproductionofmucosal waveandvoice.
When the cover and the ligament of the three types ofGAGs(chondroitin,heparananddermatansulfate)were compared separately,there wasnosignificant difference. However,bothonthecoverandtheligament,the compar-isonbetweenthe typesofGAGsshows significantlyhigher concentrationofdermatansulfate(Tables4and5).Pawlak etal.2carriedoutastudyonthepresenceanddistributionof
at0---3+.Theauthorsobservedkeratansulfateanddecorin (whichcanbindcovalentlytoQSorDS)infibrillar compo-nentsintheLP.Decorinpredominatedinthesurfacelayer (2+)inrelationtothedeeplayersandQSinthedeepones relativetothesurface.CSwasobservedonlyintheLPcell cytoplasm(2+).HSPGwereobservedinthebasallaminaand bloodvessels,aswellasinthecytoplasmofmacrophages, andprobablyfibroblasts(2+).Giventheresults,theauthors point to the probable importance of fibromodulin (cova-lentlybound to QSin the deeplayers and decorin in the surface,bothrelatedtotheregulationofthetypeandsize oftheproducedcollagen.TheauthorsdidnotstudyDS,the GAGfoundin greaterconcentrationinthisstudy.Its pres-encein large amountsin thetwolayers ofthe vocal fold shouldbebetterunderstoodaswellasthehigheramountof GAGsobservedwithaging.
Data from this study point to the need for further researchwithlargernumbersofindividualsandnotjust sul-fatedGAGs,butalsothedifferenttypesofPGsformedfrom itsconnectiontothedifferentproteinstructures,to under-standthefunctionofeachGAGandPGinthephysiologyof thevocalfoldasasoundsource.
Conclusions
1. Dermatansulfate,chondroitinsulfate andheparan sul-fatewereobservedinthecoverandligamentofhuman vocal folds,of both genders,and theconcentrationof dermatansulfateissignificantlyhigherinalllayers. 2. Therewerenosignificantdifferencesinthe
concentra-tionsofGAGsbetweencoverageandligamentaccording togender.
3. The concentration of GAG in the cover is significantly lower in individuals below 60 years compared to the elderly.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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