w w w . r b o . o r g . b r
Original
Article
Profile
of
collagen
gene
expression
in
the
glenohumeral
capsule
of
patients
with
traumatic
anterior
instability
of
the
shoulder
夽
,
夽夽
Paulo
Santoro
Belangero
a,∗,
Mariana
Ferreira
Leal
a,b,
Alberto
de
Castro
Pochini
a,
Carlos
Vicente
Andreoli
a,
Benno
Ejnisman
a,
Moises
Cohen
aaDepartmentofOrthopedicsandTraumatology,FederalUniversityofSãoPaulo(Unifesp),SãoPaulo,SP,Brazil
bDepartmentofMorphologyandGenetics,FederalUniversityofSãoPaulo(Unifesp),SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received22July2013 Accepted21October2013 Availableonline23October2014
Keywords:
Shoulderinstability Jointcapsule Geneexpression Extracellularmatrix Collagen
a
b
s
t
r
a
c
t
Objective:ToevaluatetheexpressionofthegenesCOL1A1,COL1A2,COL3A1andCOL5A1in
theglenohumeralcapsuleofpatientswithtraumaticanteriorinstabilityoftheshoulder.
Methods:Samplesfromtheglenohumeralcapsuleof18patientswithtraumaticanterior
instabilityoftheshoulderwereevaluated.Malepatientswitha positivegriptestanda Bankartlesionseenonmagneticresonanceimagingwereincluded.Allthepatientshad sufferedmorethanoneepisodeofshoulderdislocation.Sampleswerecollectedfromthe injuredglenohumeralcapsule(anteroinferiorregion)andfromthemacroscopically unaf-fectedregion(anterosuperiorregion)ofeachpatient.Theexpressionofcollagengeneswas evaluatedusingthepolymerasechainreactionafterreversetranscriptionwithquantitative analysis(qRT-PCR).
Results:TheexpressionofCOL1A1,COL1A2andCOL3A1didnot differbetweenthetwo
regionsoftheshouldercapsule.However,itwasobservedthattheexpressionofCOL5A1
wassignificantly lower in the anteroinferior region than in the anterosuperiorregion (median±interquartilerange:0.057±0.052vs.0.155±0.398;p=0.028)oftheglenohumeral capsule.
Conclusion:Theaffectedregionoftheglenohumeralcapsuleinpatientswithshoulder
insta-bilitypresentedreducedexpressionofCOL5A1.
©2014SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditora Ltda.Allrightsreserved.
夽
Pleasecitethisarticleas:BelangeroPS,LealMF,deCastroPochiniA,AndreoliCV,EjnismanB,CohenM.Perfildeexpressãodegenes docolágenonacápsulaglenoumeraldepacientescominstabilidadetraumáticaanteriordoombro.RevBrasOrtop.2014;49:642–646.
夽夽
WorkdevelopedintheDisciplineofGeneticsandtheDisciplineofExerciseandPhysicalActivityMedicineoftheDepartmentof OrthopedicsandTraumatology,FederalUniversityofSãoPaulo,SãoPaulo,SP,Brazil.
∗ Correspondingauthor.
E-mail:psbelangero@gmail.com(P.S.Belangero).
http://dx.doi.org/10.1016/j.rboe.2014.10.008
Perfil
de
expressão
de
genes
do
colágeno
na
cápsula
glenoumeral
de
pacientes
com
instabilidade
traumática
anterior
do
ombro
Palavras-chave:
Instabilidadedoombro Cápsulaarticular Expressãogênica Matrizextracelular Colágeno
r
e
s
u
m
o
Objetivo: Avaliara expressãodosgenesCOL1A1,COL1A2,COL3A1eCOL5A1nacápsula
glenoumeraldepacientescominstabilidadeanteriortraumáticadoombro.
Métodos: Foramavaliadasamostrasdecápsulaglenoumeralde18pacientescom
insta-bilidadeanteriortraumáticadoombro.Foramincluídospacientesmasculinos,comteste deapreensãopositivoelesãodeBankartnoexamederessonânciamagnética.Todosos pacientessofrerammaisdeumepisódiodeluxac¸ãodoombro.Foramcoletadasamostras dacápsulaglenoumerallesionada(regiãoanteroinferior)edaregiãomacroscopicamente nãoafetada(regiãoanterossuperior)decadapaciente.Aexpressãodosgenesdecolágeno foiavaliadaporreac¸ãoemcadeiadapolimeraseapóstranscric¸ãoreversacomanálise quan-titativa(qRT-PCR).
Resultados: AexpressãodeCOL1A1,COL1A2eCOL3A1nãodiferiuentreasduasregiõesda
cápsuladoombro.Noentanto,foiobservadoqueaexpressãodeCOL5A1estava significan-tementereduzidanaregiãoanteroinferioremrelac¸ãoàregiãoanterossuperior(mediana± intervalointerquartílico:0,057±0,052vs0,155±0,398;p=0,028)dacápsulaglenoumeral.
Conclusão: Aregiãoafetadada cápsulaglenoumeralde pacientescominstabilidadedo
ombroapresentouumaexpressãoreduzidadeCOL5A1.
©2014SociedadeBrasileiradeOrtopediaeTraumatologia.PublicadoporElsevier EditoraLtda.Todososdireitosreservados.
Introduction
Thegreatrangeofmotionprovidedbythescapularbeltallows
the glenohumeral joint tobe used as astable fulcrum for
placing the extremities ofthe upper limbsin a variety of
spatial positions. However, one consequence of this great
rangeofmotionisthatthisjointhasapropensitytobecome unstable.1
Itisbelievedthattheshoulderisthejointofthehuman bodythatmostfrequentlysuffers dislocation,withan inci-dence of8.2–23.9cases per 100,000 individuals per year.2,3 Amongthesecases,95%arecausedbytraumaticeventsand lesionsoftheanteriorcapsuleareinvolvedin90%ofthese individuals.4,5Episodesofshoulderdislocationoccurmost fre-quentlyinyoungmaleindividuals.6Manyoftheindividuals affectedpracticecompetitivesports.7Therecurrenceratefor
shoulderdislocationishighandreachesupto100%among
youngathletes.8,9
Theanteroinferior (AI) region ofthe glenohumeral cap-suleis thelocation mostaffected inepisodes oftraumatic shoulder dislocation.10 After the first episode of
ante-rior shoulder dislocation, it is common for patients to
presentshoulderinstability.8,9Patientswithshoulder insta-bilitygenerally present plastic deformationof the capsule,
which may result in capsule laxity.10,11 Previous
stud-ies have demonstrated that plastic deformation of the
capsule is necessary even in the first dislocation.12–14
Currently, little is known about the structure of the
capsule, especially among patients with shoulder
insta-bility. Better comprehension of the underlying biology is
importantfor guiding patient management and for
devel-oping newtherapeutic options that are complementary to
surgery.
Thecapsuleiscomposedofcellularandfibrouselements. Thecollagencontentofthecapsuleprogressivelyincreases duringembryonicdevelopmentandatbirththistissueis gen-erally fibrous.15 Types I, III and V fibrillar collagenare the
commonest types in the shoulder capsule.16 Mutations in
the genes that code forthese collagens have been
identi-fiedinmostofthe formsofEhlers–Danlossyndrome(EDS)
andimperfectosteogenesis,17,18whichpresentfrequent dis-locationsinseveraljoints,includingtheshoulderjoint.Thus, alterations tothesegenes mayalsoplayaroleinshoulder instability.
Theaimofthepresent study wastocompare the
mes-sengerRNA(mRNA)expressionofCOL1A1,COL1A2,COL3A1
andCOL5A1betweenaninjuredregionandanother,uninjured
regionoftheglenohumeralcapsuleinpatientswithtraumatic anteriorshoulderinstability.
Materials
and
methods
Patients
Samples were collected from the glenohumeral capsuleof
18patientswithtraumaticanteriorshoulderinstabilitywho
underwent arthroscopic surgicaltreatment atHospital São
Paulo,FederalUniversityofSãoPaulo(UNIFESP),betweenJune 2011andJune2013.Duringjointpropaedeutics,anypresence ofassociatedlesionswas noted.Allthepatientswere seen topresentcapsuleredundancyinthe anteroinferiorregion.
Afreeandinformed consentstatementwasobtainedfrom
ageatthetimeofthefirstepisodeofdislocationhadbeen25 years(range:14–37).Themeantimethatelapsedbetweenthe firstdislocationandsamplecollectionwas5years(range:4 months–10years).
OnlypatientswithapositivegriptestandaBankartlesion
shown on magnetic resonance imaging examination were
includedinthestudy.Inaddition, allthepatientsreported thattheyhadhadtwoormoreepisodesofshoulder disloca-tion.Toreducetheheterogeneityofthe sample,onlymale individualswereincluded.Afterthefirstepisodeof disloca-tion,thepatientsweretreatedwithshoulderimmobilization foratleasttwoweeks.Noneofthepatientshadanyhistoryof previoussurgeryrelatingtoshoulderinjuries.
Patientswithclinicalsignsofposteriorand/or multidirec-tionalinstabilityandthosewithgeneralizedhypermobilityor hyperlaxityaccordingtoBeighton’sscalewereexcludedfrom thestudy.19
Tissuesamples
Tissuesampleswerecollected fromtworegionsofthe cap-suleineachpatient:onefromtheinjuredregion(AIregion) andtheotherfromthecorrespondingmacroscopically unaf-fectedregion(control),i.e.theanterosuperior(AS)region.All thesampleswereimmediatelyimmersedinRNAlater® solu-tion(Qiagen,Germany)andwerethenstoredat−20◦Cuntil thetimeofRNAextraction.
Analysisofgeneexpression
TheRNAextractionwasperformedusingtheRNeasy® mini-kit (Qiagen,Germany).Theconcentration andquality were
determined usinga NanoDrop® spectrophotometer (Kisker,
Germany)andtheintegritywasascertainedbymeansof elec-trophoresison1%agarosegel.Synthesisofcomplementary
DNA (cDNA) wasperformed using the high-capacity cDNA
archivekit(LifeTechnologies,USA).
TheexpressionofCOL1A1, COL1A2, COL3A1and COL5A1
was evaluated by means of the polymerase chain
reac-tion after reverse transcription with quantitative analysis
(qRT-PCR), using the 7500 Fast Real-Time PCR system
(Life Technologies, USA). Os genes ACTB and GAPDH were
selected as internal controls for normalizing the initial
quantity of cDNA and correcting possible variations that
might affect the efficiency of the qRT-PCR. All qRT-PCR
runs were performed in triplicate for all the target genes
(COL1A1: Hs00164004mL;COL1A2:Hs00164099mL;COL3A1:
Hs00943809mL;andCOL5A1:Hs00609088mL)andforthe ref-erencegenes(-actin,ACTB:4352935e;and
glyceraldehyde-3-phosphatedehydrogenase,GAPDH:Hs99999905mL)through
usageofcommerciallyavailableprimersandprobes(Life Tech-nologies,USA).
Thegeneexpressionwasdeterminedinaccordancewith
the formula: Ct (cycle threshold)=Ct target gene (colla-gen)−geometric mean of the Ct ofthe reference genes.20
The quantification of the expression was adjusted for
the amplification efficiency of each gene (COL1A1=92%;
COL1A2=97%;COL3A1=103%;COL5A1=94%;ACTB=91%;and
GAPDH=92%).
10
5
0
−5
−10
−25
−50
COL1A1
Fold - change
COL1A2 COL3A1 COL5A1
Fig.1–Alterationofcollagengeneexpressioninthe glenohumeralcapsuleofpatientswithshoulderinstability. Expressionvaluesintheanteroinferiorregioninrelationto thecorrespondingvaluesintheanterosuperiorregionof theglenohumeralcapsule(fold-change).Horizontallines indicatethemediansandinterquartileintervals.
Statisticalanalysis
The Shapiro–Wilk testwas performed toevaluate whether
the data presentednormaldistribution. Sincethe majority of the variables analyzed didnot present normal
distribu-tion,thenonparametricWilcoxontestwasusedtocompare
geneexpressionbetweenpairsofsamplesfromthe anteroin-ferior and anterosuperior regions. The effect size for the Wilcoxontest(r)wascalculatedinaccordancewiththe for-mula r=Z/√N,21 in which r<0.1 was considered to be a trivialeffect,0.1≤r<0.3small,0.3≤r≤0.5moderateandr>0.5 large.
Spearman’scorrelationwasusedtoevaluatewhetherthere
was anycorrelation betweencollagengene expressionand
theageatwhichthepatientwasaffected,theageatthetime ofthesurgeryorthetimethathadelapsedbetweenthefirst dislocationandsamplecollection.
p values <0.05 were considered statistically significant. Mediansand interquartileintervals ofthe dataobtainedin thisstudyarepresented.
Results
TherelativeexpressionofthegenesCOL1A1,COL1A2,COL3A1
andCOL5A1presentedwidevariationamongtheindividuals
withtraumaticanteriorshoulderinstability.Bothincreased
and decreased collagen gene expressioninthe
anteroinfe-rior region, in relation to the anterosuperior region, was observed (Fig. 1). The expression of COL1A1, COL1A2 and
COL3A1 didnot differsignificantly betweenthe AI and AS
regions(Table1).However,theAIregionpresentedreduced
expression of COL5A1 in relation of the AS region of the
glenohumeral capsule ofpatients withshoulder instability (p=0.028;r=−0.3665;Table1).
Table1–Collagengeneexpressionintheglenohumeralcapsuleofpatientswithshoulderinstability.
Gene AI(median±IQ) AS(median±IQ) pvalue Effectsize(r)
COL1A1 1.068±1.597 1.681±1.801 0.372 −0.1488
COL1A2 0.555±0.413 0.650±0.455 0.145 −0.2431
COL3A1 0.716±0.608 0.678±0.662 0.879 −0.0253
COL5A1 0.057±0.052 0.155±0.398 0.028a −0.3665
AI,samplesfromtheanteroinferiorregion;AS,samplesfromtheanterosuperiorregion;IQ,interquartileinterval.
a Statisticallysignificantdifferencebetweentheanteroinferiorandanterosuperiorregions,fromWilcoxonanalysis,p<0.05.
Discussion
Thisstudydemonstratedthatpatientswithanteriorshoulder instabilitypresentdecreasedexpressionofthegeneCOL5A1
inthe AI region ofthe glenohumeral capsule,in compari-sonwiththeAS region.TypeVcollagenaccountsfor2–5% ofthetotalamountofcollageninmosttissues.22Thistype ofcollagenintercalateswithtypeIcollagen,whichisthe pre-dominantproteininthecapsule,toformheterotypicfibrils.23 Although type V collagen is the fibrillar collagen present inthe smallest quantities,it hasa central role in regulat-ingfibrillogenesisintheconnectivetissues.24 InEDScases,
decreasedCOL5A1expressionhasbeendescribedinaround
25–30%ofthepatientswithmutationsofthisgene.25Inthese patients,formationofabnormalcollagenfibrilsthatarewide andirregularhasbeenobserved.26Inaclassicalanimalmodel
forEDS,decreasedCOL5A1expressionwas associatedwith
reductionsinthenumbersoffibrilsandwithpresenceofa verylargeandstructurallyaberrantsubpopulationoffibrils withdeficienciesoftypeVcollagen.24DeregulationofCOL5A1
expressionseemstohave afundamental role instructural
alterationstothejointcapsule,bothinindividualswithEDS
andinanimalmodelsforthis syndrome.Thus,we
hypoth-esizedthatdecreasedCOL5A1expressionintheAIregionof theglenohumeralcapsuleinpatientswithshoulderinstability couldresultindisorganizedcollagenfibrils,thereby contribut-ingtowardsincreasedlaxityofthistissueintheindividuals evaluated.14,27Ontheotherhand,decreasedCOL5A1 expres-sionintheAIregioninrelationtotheASregionmightalso beanintrinsiccharacteristicoftheAIregionthatwould con-tributetowardsgreatersusceptibilityofthisregiontoinjury, inthepatientsinvestigated.
TheexpressionofthegenesCOL1A1,COL1A2andCOL3A1
didnotdifferbetweenthe AIand ASregions. However,the
number ofsamples evaluated was a limiting factor in the
presentstudy.Withthenumberofsamplesobtained,our anal-ysishadapowerofaround80%fordetectingavariationwith aneffectsizeof75%(largeeffectsize),foratypeIerrorof 5%.Largeeffectsizeswerenotdetectedinanyofthe
anal-yses and thereforethe observedpower waslow. Thus,our
analyseshadahighlikelihoodofnotdetectingadifference betweenthegroupsthatinrealityexisted(false negatives). Furtherstudiesareneededinordertounderstandtheroleof
COL1A1,COL1A2andCOL3A1intheglenohumeralcapsuleof
patientswithshoulderinstability.
Inthepresentstudy,onlymalepatientswhohadhadmore thanoneepisodeofshoulderdislocationwereevaluated.The aiminmakingthisselectionwastohomogenizeoursample.
Inaddition,pairedsampleswithlesionsandwithoutlesions (controls)wereobtainedfromtheglenohumeralcapsuleofthe sameindividual,soastoavoidthepossibilityofbiascaused bybiologicalvariationsbetweenindividuals.Thisisastrategy commonlyusedinmolecularstudiesthatseektounderstand theprocessesofcarcinogenesis.28However,themRNAlevels ofallthecollagengenesstudiedwereheterogenousbetween thepatientswithshoulderinstability.Thisheterogeneitymay
have been due todifferent environmental exposures,such
as variationsinthenumber ofepisodes ofdislocation and
the time that elapsed between the dislocating events and
the surgery. Moreover, like other acute soft-tissue injuries, shoulderinstabilityisamultifactorialdiseaseandtherefore intrinsicfactors,suchaspolymorphismsofcollagengenes29,30 or ofgenescodingforproteinsinvolvedinregulating their
expression, may also contribute towards heterogeneity
betweenpatientsandtowardsgreaterriskofthedisease. Because the onlytissuesamples studiedwere collected duringthesurgicaltreatment,it wasnotpossibletoassess theregulatorydynamicsofgeneexpression.Nonetheless,it shouldbenotedthatthisstudy,tothebestofourknowledge, wasthefirsttoevaluategeneexpressionintheglenohumeral capsuleofpatientswithshoulderinstability.Thisstudythus contributestowardscomprehendingthiscondition.
Conclusions
Theinjuredregionoftheglenohumeralcapsuleofpatients withshoulderinstabilitypresenteddecreasedCOL5A1 expres-sion.Studyinggeneexpressionintheglenohumeralcapsule ofpatientswithshoulderinstabilityisnovelintheliterature andopensupnewresearchperspectivesregardingthis con-dition.Evaluationofgeneexpressionmaycontributetowards greatercomprehensionofthebiologyoftheselesionsandthus towardsdevelopmentofnewtreatmentstrategies.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
WethanktheNationalCouncilforScientificandTechnological
Development(CNPq)andtheResearchSupportFoundationof
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