centrifugedwww.diediao.com

EDITORIALChronicrenalfailure,vascularcalcificationandtheRNK/RANKL/OPGsystemOlmosJM,HernándezJLORIGINALSEffectofRANK/RANKL/OPGpathwayonbonedemineralizationandvascularcalcificationinchronickidneydiseaseMartínezAriasL,SolacheBerrocalG,PanizoGarcíaS,CarrilloLópezN,AvelloLlanoN,QuirósCasoC,NavesDíazM,CannataAndíaJBTheassociationofMMP11G>2GpolymorphismwithaorticvalvecalcificationSolache‐BerrocalG,BarralA,MartínM,Román‐GarcíaP,LlosaJC,Naves‐DíazM,Cannata‐AndíaJB,RodríguezIFunctionalstudyofpromotergenepolymorphismsofsclerostinPérez‐CampoFM,SaudoC,KrebesovaR,Delgado‐CalleJ,RianchoJAPrevalenceoflowlevelsofvitaminDinpatientswithbreastcancerwholiveinNorthernlatitudes21-22González‐FisherRF,Pérez‐JaimeS,BuzK,Sotelo‐FélixE,lvarezOrdoricaO,GonzálezRiestraHJ,RolonPadillaACLINICALNOTEHemochromatosisandosteoporosis,inreferenceto4casesMontaoJaramilloD,DíazCurielMREVIEWOxidativestressasapossibletherapeutictargetforosteoporosisassociatedwithagingPortal‐NúezS,delaFuenteM,DíezA,EsbritP101105115121127134138SUMMARYVol.
8-N4-October-December2016OurcoverProstatecancer.
MetastasisinavertebralbodyAutor:CourtesyofProfessorAlanBoyde.
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PhDDepartmentofMedicine,DivisionofEndocrinology.
IndianaUniversitySchoolofMedicine.
Indianapolis,Indiana.
EstadosUnidosErnestoCanalis.
MD,PhDDirector,CenterforSkeletalResearch.
ProfessorofOrthopedicSurgeryandMedicineNewEnglandMusculoskeletalInstituteUniversityofConnecticutHealthCenter.
Farmington,CT.
EstadosUnidosDr.
OswaldoDanielMessinaFacultaddeMedicina.
UniversidaddeBuenosAires.
HospitalCosmeArgerich.
BuenosAires.
ArgentinaPatriciaClarkPeralta.
MD,PhDFacultaddeMedicina,UNAM.
UnidadClínicaEpidemiológica.
HospitalInfantilFedericoGómez.
MéxicoDF.
MéxicoDr.
CarlosMautalenProfesorConsultorTitulardelaFacultaddeMedicina.
UniversidaddeBuenosAires.
Directorde"Mautalen,SaludeInvestigación".
BuenosAires.
Argentina.
LilianIPlotkin.
PhDAnatomyandCellBiology.
IndianaUniversitySchoolofMedicine.
Indianapolis,Indiana.
EstadosUnidosDr.
ManuelDíazCurielUniversidadAutónomadeMadrid.
UnidaddeMetabolismoseo.
HospitalFundaciónJiménezDíaz.
InstitutodeInvestigaciónFJD.
FundaciónHispanadeOsteoporosisyMetabolismoMineral(FHO-EMO).
Madrid.
EspaaDr.
AdolfoDíezPérezUniversidaddeBarcelona.
ServiciodeMedicinaInterna.
InstitutoMunicipaldeInvestigaciónMédica.
(IMIM).
HospitaldelMar.
Barcelona.
EspaaDr.
JosepBlanchRubióServiciodeReumatología.
HospitaldelMar,Barcelona.
InstitutoMunicipaldeInvestigacionesMédicasdeBarcelona.
ParquedeInvestigaciónBiomédicadeBarcelona.
EspaaDr.
ManuelSosaHenríquez(Director)UniversidaddeLasPalmasdeGranCanaria.
GrupodeInvestigaciónenOsteoporosisyMetabolismoMineral.
HospitalUniversitarioInsular.
ServiciodeMedicinaInterna.
UnidadMetabólicasea.
LasPalmasdeGranCanaria.
EspaaDra.
MaríaJesúsGómezdeTejadaRomero(Editor)UniversidaddeSevilla.
DepartamentodeMedicina.
Sevilla.
Espaa100COMMITTEESS/RevOsteoporosMetabMiner.
2016;8(4):100ReviewersVolume8(2016)TheBoardandtheDirectorateSEIOMMMagazinethanksyouforyourinvaluableassistance.
MJoséAmérigoGarcíaJosepBlanchiRubióJoséRamónCaeiroReyJavierCalvoCataláAntonioCanoSánchezCristinaCarbonellAbellaEnriqueCasadoBurgosBernardinoDíazLópezAdolfoDíezPérezJesúsDelgadoCalleCasimiraDomínguezCabreraJoséFilgueiraRubioJordiFiterAresteCarlosGómezAlonsoMJesúsGómezdeTejadaRomeroJesúsGonzálezMacíasEmilioGonzálezReimersDanielGrinbergVaismanNuriaGuaabensGayDiegoHernándezHernándezJoséLuisHernándezHernándezGabrielHerrero-BeaumontCuencaJorgeMaloufSierraMElenaMartínezRodríguezMJoséMontoyaGarcíaLauraNavarroCasadoXavierNoguésiSolánSantiagoPalaciosGil-AntuanoJoséLuisPérezCastrillónConchadelaPiedraGordoLilianPlotkinJoséManuelQuesadaGómezLuisdelRíoBarqueroMinervaRodríguezGarcíaManuelSosaHenríquezCarmenValdésyLlorcaCarmenValeroDíazdelaMadrid101EDITORIAL/RevOsteoporosMetabMiner.
2016;8(4):101-104OlmosJM*,HernándezJLDepartamentodeMedicinaInterna-HospitalUniversitarioMarquésdeValdecilla-IDIVAL-UniversidaddeCantabria-Santander(Spain)Chronicrenalfailure,vascularcalcificationandtheRNK/RANKL/OPGsystemardiovascularcomplicationsareamongthemostimportantclinicalchallengesinpatientswithchronickidneyfailure(CKF).
Thesearefrequentprocessesthatpresenthighmorbidityandmorta-lity.
Asanexample,around50%ofpatientswithterminalCRFdiefromthisdisease1.
Renalpatientspresenttwotypesofvascularcalcifi-cations:calcificationofthetunicamedia,alsocalledMnckebergsclerosis,inwhichthemineralisdepositedwithinthelayerofsmoothmuscle.
Thesecondtypeiscalcificationoftheintima,inwhichthecalciumdepositoccursaftertheaccumulationofcholesterolunderthedamagedendothelialmonolayer2.
Calcificationofthetunicamedia,wherevascularsmoothmusclecells(VSMC)andelasticfibersarefound,isnotrelatedtocholesterollevelsortheexistenceofatheromatousplaquesandcausesthehardeninganddecreaseinthearte-ries'distensibility.
AtheroscleroticcalcificationoftheintimamayalsooccurinpatientswithCRF.
Inthesecases,intimalcalcificationisassociatedwiththesubintimaldepositoflipidsandlipoproteins,whichmaystimulatethedevelopmentofimmuneresponses,bothinnateandadaptive,inducingendothelialcellsandtheVSMCtoexpressinflam-matorymolecules,whichstimulatetumor-infiltra-tingmonocyte/macrophage.
Asaresult,increasedinflammation,oxidizedlipidsandfibrousmatrixsecretioninatheroscleroticlesionsfurtheracceleratevascularcalcification,whicheventuallyleadstoatheroscleroticplaquerupture1-3.
InpatientswithCKF,bothatheroscleroticintimalcalcificationandtunicamediacalcification,independentofatherosclerosis,areassociatedwithanincreaseincardiovascularmortalitycomparedtopatientswithCKFwhodonotpresentit4.
Initiallythiswasconsideredasecondarydisordertothepassivedepositofcalciumandphosphorusinthevascularwall.
However,morerecentlyvas-cularcalcificationhasbeenfoundtobeaperfectlyregulatedprocessbywhichVSMCundergomole-cularandphenotypicchanges.
Withthesealtera-tionstheyacquiresomeofthefunctionsthatcha-racterizeosteo-chondrocitarystraincells1,2,andleadtothereleasebyvesicularVSMCstructurescontaininghydroxyapatite5.
Inthisprocessof"osteo-chondrocytetransdifferentiation"differentfactorsinvolvedinthedifferentiationofbonecells,suchasRunx2,bonemorphogenicproteins(BMPs),RANK/RANKL/OPGsystemorWntpath-waywouldintervene.
Furthermore,inpatientswithCKFandinanimalmodelsofthisdisease,increasedvascularcalcificationisaccompaniedbyareductioninbonemass,suggestingthatthesig-nalsinvolvedinboneandvascularwallminerali-zationmaybehavedifferentlydependingonthetissuemicroenvironmentinwhichtheyact6,7.
CKFischaracterizedbychangesinbonemetabo-lismthat,inadditiontobeingdetrimentaltotheskeleton-renalosteodystrophy-favorcalcificationofsofttissuesandvessels.
Hypercalcemiaandhyperphosphatemia,hyperparathyroidism,increa-sedfibroblastgrowthfactor23(FGF23),increasedoxidativestressanddecreasedinhibitorsofcalci-ficationsuchasfetuin-Aandpyrophosphatescouldallplayaroleinthevascularcalcificationprocess1,2,6,7.
Hyperphosphatemia,aswellashypercalcemia,aretwoofthemainfactorsassociatedwiththedeve-lopmentofvascularcalcificationinCRF8.
Thedietwithhighphosphoruscontentincreasesvascularcalcificationandreducesbonemassinratswithchronicrenalfailure.
Ontheotherhand,treat-mentswithhighcalciumand/orphosphatecontentinducethecalcificationofVSMCinexperimentalanimals.
Althoughthemechanismsinvolvedinthisprocesshavenotyetbeenaccuratelyfound,hyperphosphatemiahasbeenshowntoinducevascularcalcification,favoringosteogenicexpres-sionsuchasRunx2orBMP29,10.
Someauthorshaveshownthat,unlikenormalvessels,thearteriesofCKDpatientsexpressRunx21,2,9,10.
Ontheotherhand,theuremicserumincreasestheexpressionofRunx2andthecalcificationoftheVSMC.
Inaddition,hyperphosphatemiaactivatestheWntpathway,favoringβ-catenintranslocationinthesmoothmusclecellnucleus,therebystimulatingtheexpressionofdirecttargetgenessuchascyclinD1,axin2andVCAN/versican10.
Finally,hyper-phosphatemiaalsoincreasesthelevelsofFGF23,which,togetherwithitsco-receptorklotho,mayplayapathogenicroleinarterialcalcificationandinthealterationofskeletalmineralization11.
TheroleofPTHisalsocomplex.
Inhemodialysispatients,increasedPTHisassociatedwithvascu-larcalcificationand,inratswithrenalfailure,bothaorticcalcificationandlossofbonemassareasso-*e-mail:miromj@humv.
esCDOI:http://dx.
doi.
org/10.
4321/S1889-836X2016000400001102EDITORIAL/RevOsteoporosMetabMiner.
2016;8(4):101-104ciatedwithincreasedphosphorusandPTH12.
However,inotherstudiesithasbeenpointedoutthatPTHisnotabletodirectlyinducevascularcalcification,butwouldhaveasynergisticeffectwithphosphate,whichwouldberelatedtoincre-asedosteoclasticactivityandboneremodelingthatthishormonedetermines.
Thisincreaseinboneremodelingfavorscalciumandphosphoruslossfromthebone,thusstimulatingvascularcal-cification.
Itisoneofthedeterminantsofthemostfrequentformsofrenalosteodystrophy,osteo-pathywithhighremodelingorsecondaryhyper-parathyroidism.
Atothertimes,aswithadynamicbone,thelowboneremodelingdeterminesanalterationinboneformationandmineralization,withtheconsequentreduceduseofexcesscal-ciumandphosphorus,whichalsofavorsvascularcalcification13,14.
TheincreaseinoxidativestressobservedinpatientswithCRFwouldalsobecloselyassocia-tedwiththedevelopmentofvascularcalcification.
Aswithhyperphosphatemia,thiseffectwouldbemediatedthroughtheexpressionofRunx2intheVSMC15.
Inaddition,arecentstudycarriedoutinpostmenopausalwomenfoundthatincreasedoxi-dativestresswasassociatedwithanincreasedriskofhipfracture,suggestingthattherewouldbeaninverserelationshipbetweenoxidativestressandmineralmetabolism1,5.
Alongwithincreasedlevelsofcalciumandphos-phorus,thedecreaseinsomeoftheinhibitorsofcalcification,suchasfetuin-Aandpyrophosphate,whichcanbeobserved,cancontributetotheincre-aseofvascularcalcificationinthesepatients1,2,6.
InthisissueoftheJournalofOsteoporosisandMineralMetabolism,MartínezAriasetal.
16analy-zedtheeffectsoftheRANK/RANKL/OPGsystemonbonedemineralizationandvascularcalcifica-tioninCRF.
Theseauthorsuseinvivoandinvitromodelsofvascularcalcificationtoverifythatratswithchronicrenalfailureandadiethighinphos-phoruspresentdecreasedbonemineraldensity,togetherwithaorticcalcificationsthatareaccom-paniedbyanincreaseinRANKLgeneexpressionandadecreaseinOPG.
Inthetibiaoftheseani-malsbothRANKLandOPGexpressionincreased,althoughtheincreaseinOPGoccurredatearlierstages.
IntheVCAM,theadditionofuremicserumandcalcifyingmediuminducedanincreaseincal-ciumcontentandRANKLandOPGexpression,whiletheadditionofOPGandthesilencingofRANKinhibitedthisphenomenon.
Therefore,theseauthors'resultsconfirmtheRANK/RANKL/OPGaxisinvolvementinthevas-cularcalcificationprocessandprobablyalsointhelossofbonemassthataccompaniesCRF.
Thisopensthedoortonewresearchlinesinthisarea.
Astheauthors16comment,thereisagreatdealofscientificevidencelinkingtheRANK/RANKL/OPGsystemtovascularcalcifications1,2,7,16-20.
ThefirstderivativeoftheOPG-nullmousestudyconduc-tedafewyearsagobyBucayetal.
17,whodemonstratedthatOPG-deficientmiceexhibitedvascularcalcifications,aswellasanintensedecre-aseinbonemineraldensity(BMD)andoneHighincidenceoffractures.
Itwaslaterfoundthattre-atmentwithrecombinantOPGsignificantlyredu-cedvascularcalcificationinmicedeficientinLDLreceptors18.
Ontheotherhand,thestudiescarriedoutinpatientswithCKDindicatethatthelevelsofRANKLandOPGincreaseasdothoseofPTHandphosphate,andithasbeenpointedoutthattheincreaseofRunx2increasestheexpressionofRANKLinVCAM.
Inanimalmodels,increasedRANKLinducesalossofbonemassandvascularcalcification,whiletheadditionofOPGhastheoppositeeffect.
ThepathwaybywhichRAKLwouldpromotecalcificationwouldbethroughbindingtoitsRANKreceptor,withtheconsequentactivationoftheNF-kBalternativepathwayandthebonemorphogenicproteins2and4(BMP2andBMP4),favoringtheosteogenictransitionoftheVSMC1,2,19,20.
Ontheotherhand,RANKLcouldalsoactindirectlybystimulatingthereleaseofpro-cytokinesbymacrophages.
Finally,andasmightbeexpected,theWntpath-wayalsoappearstobeinvolvedinthisprocess.
Wehavealreadycommentedthathyperphospha-temiawouldactivatethispathwayintheVCAM10.
Ontheotherhand,theexpressionofsclerostinincreasesinarterieswithvascularcalcification.
LevelsofsclerostinandotherWntpathwayinhibi-tors,suchasDickkopf-1(DKK1)orsolublefrizz-ledreceptor(SFR),increaseasrenalfunctiondete-rioratesandcorrelateinverselywithhistologicalparametersofboneremodelingandwiththenum-berandfunctionofosteoblasts21,22.
IthasrecentlybeenpointedoutthattheincreaseofFGF23,whichaccompaniesrenalfunctiondeterioration,couldalsoacttoinhibitthissystem11.
Therefore,sclerostinandotherinhibitorsoftheWntsystem,releasedintothemediumfromthevessels,couldacttoimpairthebonestructureandretardthemineralizationprocess.
Thesealterations,alongwiththoseoftheRANK/RANKL/OPGsystem,hyperphosphatemiaandotherfactorsdiscussedhere,couldhelpmedicalresearcherstounders-tandthecomplexrelationshipbetweenvascularcalcificationandbonelossandincreasedfracturesinpatientsWithCRF(Figure1).
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OxidativestressPi/Ca;PRH/FGF23↑↑↑FetuinA/Pyrophosphates↑BONEFORMATIONVASCULARCALCIFICATIONBONERESORTIONRunx2↑RANKL↑↑PTH↑FGF23↑+Ca+PiMoVSMCsOCCSclerostinTransdifferentiationOsteo-chondrogenicWnt↑Figure1.
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2016;8(4):105-114105MartínezAriasL1,SolacheBerrocalG1,PanizoGarcíaS1,CarrilloLópezN1,AvelloLlanoN2,QuirósCasoC2,NavesDíazM1,CannataAndíaJB11ServiciodeMetabolismoseoyMineral-InstitutoReinaSofíadeInvestigaciónNefrológica-ReddeInvestigaciónRenal(REDinREN)delInstitutodeSaludCarlosIII-UniversidaddeOviedo-Oviedo(Espaa)2LaboratoriodeMedicina-HospitalUniversitarioCentraldeAsturias-Oviedo(Espaa)EffectofRANK/RANKL/OPGpathwayonbonedemineralizationandvascularcalcificationinchronickidneydiseaseCorrespondence:ManuelNavesDíaz-ServiciodeMetabolismoseoyMineral-HospitalUniversitarioCentraldeAsturias-EdificioFINBA,PlantaprimeraF1.
1(Aula14)-AvenidadeRoma,s/n-33011Oviedo(Spain)e-mail:manuel@hca.
esDateofreceipt:07/10/2016Dateofacceptance:18/10/2016WorkawardedascholarshipResearchAMGEN-SEIOMM2010.
SummaryIntroduction:Incasesofchronickidneydisease(CKD),boneandmineralmetabolismchangesoccurwhichfavorsofttissuecalcification.
AlterationsintheRANK/RANKL/OPGsystemcouldalsofavorvascu-larcalcification,amajorcauseofmorbidityandmortalityinCKD.
Objective:Inaninvivoexperimentalmodelofchronicrenalfailureprogression,weassesstheeffectofCKDonvascularcalcificationandbonelosscorrelatingthesechangesintheRANK/RANKL/OPGpath-way.
Aninvitrosystemwasusedtoconfirmfindings.
MaterialandMethods:Twomodelsofvascularcalcificationwereused:aninvivoratmodelwithchronicrenalfailurefedonadietwithdifferentphosphoruscontent,andaninvitromodelinvascularsmoothmusclecells(VSMC)subjectedtodifferentcalcifyingstimuli.
Results:At20weeks,50%ofanimalswithadiethighinphosphoruspresentedaorticcalcificationaccom-paniedbyincreasedaorticexpressionofRANKL.
Incontrast,OPGdecreasedprobablyasaconsequen-ceofaninflammatorycomponent.
At20weeks,expressionofRANKLandOPGinthetibiaincreased,whiletheincreaseinOPGoccurredatearlierstages.
InVSMC,theadditionofuremicserumandcalcificationmediumincreasedcalciumcontentandexpres-sionofRANKLandOPG.
TheadditionofOPGandsilencingofRANKinhibitedthisincrease.
Conclusions:OurresultsconfirmRANK/RANKL/OPGsysteminvolvementinthevascularcalcificationprocess.
Keywords:RANK,RANKL,OPG,chronickidneydisease,vascularcalcification.
DOI:http://dx.
doi.
org/10.
4321/S1889-836X2016000400002ORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):105-114106IntroductionVascularcalcificationisaprocessinwhichvascu-larsmoothmusclecells(VSMC)andotherpopula-tionsofbloodvesselcellsundergoatransforma-tionandbegintoresembleosteoblasts1.
Thispro-cessisregulatedinamannersimilartobonemineralization,withseveralboneproteinsbeingimplicated2-4.
Osteoblastsarecellsresponsiblefortheformationofbonethatalsoregulatetheacti-vityofosteoclastsandthereforeplayanimportantroleinthehomeostasisofcalcium(Ca)andphos-phorus(P)5.
OsteoblastssecretetheNF-κBactiva-torreceptorligand(RANKL)thatbindstoitsreceptor(RANK)inosteoclastprecursorspromo-tingformation,activationandsurvival6-7.
Inaddi-tion,osteoblastssecreteosteoprotegerin(OPG),whichactsasasolublereceptorlureofRANKLandinhibitsthebindingofthisligandtoitstrans-membranereceptorRANK.
ThereisconsiderablescientificevidencelinkingtheRANK/RANKL/OPGsystemtovascularcalcifications,whichmaybeanimportantautocrine/paracrinesysteminvolvedintheprocess.
ThepathwaybywhichRANKLpro-motescalcificationthroughbindingtoitsRANKreceptorwiththeconsequentactivationoftheNF-κBalternativepathwayandbonemorphogeneticprotein4(BMP4)8hasbeenimplicatedintheoste-ogenictransitionofVSMCs9,10.
Chronickidneydisease(CKD)ischaracterizedbychangesinboneandmineralmetabolismthatfavorthecalcificationofsofttissuesandvessels.
AlterationsinthegeneexpressionoftheRANK/RANKL/OPGsystemcouldbefavoringvas-cularcalcification,oneofthemaincausesofmor-talityinCKD.
Itisinterestingtoinvestigatethedif-ferencesintheregulationoftheRANK/RANKL/OPGsysteminboneandvesselinordertodesignstrategiesaimedatprotectingthebonewithouthavingnegativeeffectsonvascularcalcification.
Therefore,thisstudyaims:a)toevaluateinaratmodeltheeffectofCKDanddietswithdiffe-rentPcontentonvascularcalcificationquantifiedbyCacontentanalysisandbonemineraldensity(BMD),quantifiedbybonedensitometry;B)tocorrelatethesechangeswithalterationsintheRANK/RANKL/OPGsystemgeneexpressioninarteriesandbonesoftheseanimals;Andc)touseaninvitrosystemtoconfirmthefindingsfoundinvivo.
MaterialsandmethodsInvivostudies:VascularcalcificationmodelTheprotocolwasapprovedbytheUniversityofOviedo'sEthicalCommitteeofAnimalExperimentation.
ThestudywasperformedwithmaleWistarrats(n=55)at4monthsofage(350-400g).
Surgicalintervention,followinginhalationofisofluraneanesthesia,involvedinducingchronicrenalfailure(CRF)(7/8)inasinglesurgicalprocedure.
Completenephrectomyoftherightkidneyandthensubtotalnephrectomyoftheleftkidneywerecarriedoutbylateralincision.
Thisprocedurepre-servesapproximatelyonefourthoftherenalmass.
TheratswithCRFweredividedintotwogroups:one,CRFC,fedastandardrodentdietwithnor-malPcontent(0.
6%P,0.
6%Ca,and20%proteincontent,Panlab,Barcelona,Spain),andtheother,CRFP,fedadietwithhighPcontent(0.
9%P,0.
6%Ca,and20%proteincontent,Panlab).
Thestudylasted20weeks(CRF20CandCRC20P),timerequiredtoinducevascularcalcifications.
WealsoincludedaShamgroup(n=10)thatwasfolloweduptoweek20.
Intermediateevaluationswerealsoperformedthroughoutthestudy,withsacrificesat8and12weeks(CRF8C,CRI12C,CRI8PandCRI12P).
Twenty-fourhoursbeforeslaughter,theratswerehousedinmetaboliccagesandreceiveddietandwateradlibitum.
TheyweresacrificedusingCO2anesthesia,andserumsamplesweretakenforanalysis.
Fromeachrattheabdominalaortawasremoveddowntothebifurcationoftheiliaccrestsanddividedintothreeportions:thefirstfragmentwasusedfortheextractionofRNA,thesecondfragmenttodeterminetheCacontent,andthethirdfragmentwasstoredinparaffinforfutu-restudies.
Atthetimeofsacrificethetwotibiawereremoved.
Theleftwaspreservedinalcoholtomeasurebonemineraldensity(BMD).
Theremai-ningtibiawasfrozenat-80°Cuntilprocessedforthestudyofgeneexpression.
BiochemicalmarkersSerumurea,creatinine,CaandPweremeasuredusingaHitachi717multi-channelautomaticanaly-zer(BoehringerMannheim,Berlin,Germany).
Parathyroidhormone(PTH)wasmeasuredbyELISA(Immutopics,SanJuanCapristano,USA)followingthemanufacturer'sprotocol.
BonedensitometryBMDwasmeasuredintibiaatthreelevels:proxi-maloctave,seven/eighthdistalandtotaltibia,withaHologicQDR-1000dual-energydigitalradiologicaldensitometer(Hologic,Bedford,USA)equippedwithaspecificprogramforsmallani-mals.
AnalysisofaorticcalcificationCalcificationoftherats'abdominalaortawasanalyzedbytwomethods:totalCacontentandvonKossastaining.
TodeterminetotalCacontent,afragmentoftheabdominalaorta(thecmproximaltotheiliacbifurcation)washomogenizedwithanUltraturrax(OmniHT)in0.
6NHCl.
Aftershakingat4°Cfor24hoursthesampleswerecentrifuged.
TheCacontentwasdeterminedinthesupernatantbytheo-cresolphthaleincomplexonemethod(Sigma-Aldrich,St.
Louis,USA),andthepelletwasresus-pendedinlysisbuffer(125mMTrisand2%SDS,PH6.
8)forproteinextractionandquantificationbythemethodofLowry(Bio-Rad,Hercules,USA).
TheCacontentwasnormalizedbyexpressingasgCapermgprotein.
ORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):105-114107TocarryoutvonKossastaining,anotherfrag-mentoftheabdominalaortawasincludedinmethylmethacrylate(Sigma-Aldrich).
Five5mmthicksectionswereobtainedusingaPolycutSMicrotome(Reicher-Jung,Heildelberg,Germany)andstainedfollowingthevonKossamethod.
GeneexpressionstudyRNAextractionwascarriedoutbytheguanidi-nium-phenol-chloroformthiocyanatemethod.
DNAcopy(cDNA)wassynthesizedusingthehighcapacitykit(AppliedBiosystems,FosterCity,USA).
TheRANK,RANKLandOPGgeneexpres-sionwasanalyzedbyreal-timePCR(qPCR)onAppliedBiosystemsABIPrism7000equipment.
Assayon-demandassaysdesignedbyAppliedBiosystemsemployingspecificoligosandfluores-centTaqmanprobeswereusedforeachofthePCRs.
GAPDHwasusedtoquantifyandnormali-zetheexpressionoftheconstitutivegene.
Invitrostudies:Primarycultureofvascularsmoothmusclecells(VSCM)VSCMfromprimarycultureofaortaexplantsfromhealthyWistarratsat2monthsofagewasused,sacrificing12ratsandusingCO2anesthesia.
AbdominalaortaswereremovedandintroducedintocoldPBSwith100units/mLpenicillinand100mg/mLstreptomycin(BiochromAG,Berlin,Germany).
AfterwashingabundantlywithcoldPBS,theaortaswerecutlongitudinally;Theendo-theliallayerwascarefullyremovedandsubse-quentlycutintofragments(explants)of2to3mm2.
Theexplantswereplatedinsix-wellcultureplates(Sigma-Aldrich)pretreatedwithfibronectin(10mg/cm2;Sigma-Aldrich).
Oncetheexplantswereplaced,1mLofDMEM(Dulbecco'sModifiedEagleMedium,BiochromAG)supplementedwith20%fetalbovineserum(FBS)(BiochromAG)wasadded.
Themediumwasrenewedevery2days.
Whenthecellsreachedsubconfluency,thetissuefragmentswereremovedandthecellswereenzy-maticallyseparated(0.
25%trypsinand1mMEDTA).
Cellswereseededatadensityof105cellsperculturedish(Sigma-Aldrich)withDMEMsupple-mentedwithFBS(10%).
CellsobtainedbythismethodwereidentifiedasVSCMbythefollowingcriteria:(1)cellsgrowinthecharacteristicvalleyandcholinepattern;And(2)immunostainingwaspositiveforalpha-actin(mAbfromSigma-Aldrich).
Cellsbetweenpassages2and8wereused,usingthreewellsperconditionandtheexperi-mentswereperformedintriplicate.
InductionofcalcificationinVSCMInordertoanalyzetheuremia-inducedcalcifica-tionandtoknowtheimplicationoftheRANK/RANKL/OPGsystem,twodifferentcondi-tionswereused.
Forthefirstcondition,theVSCMculturesweretreatedwithDMEMsupplementedwith15%ure-micratserum(asetof8-weekCRFratseracon-taining10.
8mg/dLCa,6.
7mg/DLP,and898pg/mLPTH).
AsacontrolconditionDMEMwasusedwith15%serumfromhealthyrats(apoolofseracontaining10.
4mg/dLCa,3.
6mg/dLPand25pg/mLPTH).
Inasecondcondition,toconfirmtheeffectofP,theVSCMswereculturedwithcalcifyingmedium:DMEMF12+0.
1%bovineserumalbumin(BSA)with2mMCaand3mMP).
DMEMcontrolF12+0.
1%BSAwasusedascondition.
Inbothcases,Cadepositionwasdetermined4and8daysafteradditionofthestimuli.
TheeffectofOPG(100pM),silencingoftheRANKreceptor(increasingconcentrationsbetwe-en100pMand100nM)wastestedinVSCMinwhichcalcificationwasinducedwithDMEMF12+0.
1%BSAwith2mMCaand3MMofP.
GeneexpressionstudyWeproceededinthesamemannerasdetailedinthesectiononinvivostudies.
Lentiviralproductionandinfection/RANKsilencingbyshRNATheRANKgenewassilencedintheVSCMbysmallforksofRNA(shRNA),whichwereclonedintoalentivirus-basedvector(FSVsi).
InitwereintroducedshRNAswhosetargetwasTTAGCT-GAGGATGCTGAGGATandscramblesequences.
Allofthemwereco-transfectedwiththevirionpackagingelements(VDV-G)ina293Tcellcultu-reusingpolyethyleneimine.
Infectiousparticleswereproducedbyculturingthecells3-4daysinmediumforVSCM.
Themediumwasthencentri-fugedat1,000gfor5minandthesupernatantwasaddedtoaVSCMculture,beingreplacedbytheconventionalmediumafterovernightincubation.
Finally,theVSCMswerecollectedafter4daysandthesilencingofRANKwithqPCRandWesternBlotwaschecked.
WesternBlotAftertransfer,themembraneswereincubatedfor12hourswithanti-RANKantibodies(1:1,000,CellsSignalingTechnology,Danvers,USA),andanti-tubulin(01:10,000,Sigma-Aldrich).
BindingofthesecondaryantibodywasdetectedwiththeWesternBlotdetectionkitECLAdvance(AmershamBioscience,Buckinghamshire,UK)andtheVersaDoc4000(Bio-Rad)imagingsystemsystem.
StatisticalanalysisForthestatisticalanalysisoftheresults,theSPSS17.
0programwasused.
Inthecaseofvariableswithnormaldistribution,thecomparisonofthetreatmentgroupswasperformedusingANOVAwiththeBonferronitest.
Inthecaseofvariableswithnon-normaldistribution,theKruskal-Wallistestwasused.
Results1.
BiochemistryInthegroupsthatreceivedthedietwithahighPORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):105-114108content(CRF8P,CRF12P,CRF20P),aslightdete-riorationofrenalfunctionwasobservedwithres-pecttotheircontrols(CRF8C,CRF12C,CRF20C).
Aggravatedat20weeks(Table1).
InthehighPdietgroup,serumCasignificantlydecreasedonlyatweek20(CRP20P),whileserumPincreasedinallgroupswithahighPdiet,particularlyat20weeksoftreatment.
ParalleltoP,PTHincreasedastreatmenttimeincreased,beingstatisticallysignifi-cantfromweek12andparticularlyatweek20,whereseveresecondaryhyperparathyroidismwasobserved(Table1).
2.
DensitometricstudyAlthoughtherewasaslightdecreaseintheBMDofthegroupsofanimalswithhighdietinPregar-dingtheircontrolsinallthestudiedsectors,thiswasonlysignificantat20weeks(Table2).
Losseswerepredominantatthedistallevel,wherethereisahighercontentofcorticalbone,onlossesattheproximallevel(Table2).
3.
InvivoeffectofuremiaandPoverloadonvascularcalcification,boneactivityandRANK/RANKL-OPGsystemAlthoughtheCacontentoftheaortasofanimalsfedanormalP-contentdietwasslightlyaffectedbyuremia,administrationofadietwithhighPcontentincreasedCasignificantlyinatime-dependentmannerwithrespecttotheShamgroup.
AnimalsreceivingthehighPdietincrea-sedtheaorticcontentofCawithrespecttotheirrespectivecontrolsfromweek12,withthiseffectbeingmagnifiedatweek20.
Despitethegenera-lizedincreaseinaorticCacontent,vonKossarevealedvisiblecalcificationsintheaortainonly50%oftheanimalswithdietwithhighcontentinP(Figure1).
ParalleltotheincreaseinCacontenttherewasanelevationofRANKLexpressionintheaorta(Figure2A).
RANKexpressiondidnotshowanydifferencesalongthecourseofCRI(Figure2B),whereasOPGdecreasedinalluremicgroups,par-ticularlythosereceivingahighPdiet(Figure2C).
Inthetibia,anincreaseinRANKLandOPGexpressionwasobservedatweek20ofthehighPdietgroup(Figures3A,3C).
OPGexpressionalsoincreasedinallgroupsreceivinghighPdiet,notingtheincreaseobservedatweek20.
Incon-trast,RANKexpressionremainedsimilarinallgroups.
4.
InvitroeffectofuremiaandPoverloadonvascularcalcificationandtheRANK-RANKL-OPGsystemUremicseruminducedasignificantincreaseinCacontentat4and8days(Figure4A).
TherewasasignificantincreaseintheexpressionofRANKL(at4and8days)andOPG(at8daysoftreatment)(Figures4B,4Cand4D).
Calcifyingmedium-treatedVMCV(DMEMF12,2mMCa,3mMP)showedasignificantincreaseintime-dependentCacontent(Figure5A).
InparallelincreasedRANKLandOPGexpression(Figures5B,5Cand5D).
5.
InvitroeffectoftheadditionofOPGoncal-cificationinducedbyuremicserumToconfirmtheideathatincreasedRANKLexpres-sionisresponsiblefortheCacontentincreaseinVSCMtreatedwithuremicserum,100pMOPGaddedtotheculturemedium,whichledtoasig-nificantdecreaseofOPG(Figure6).
6.
InvitroeffectofRANKsilencingoncalcifi-cationinducedbyuremicserumSimilarly,silencingoftheRANKreceptorbytheshRNAtechniquesignificantlyreducedtheCacon-tentoftheVSCMtreatedwithuremicserum(Figure7).
DiscussionCKD,adiseasecharacterizedbyaprogressivelossofrenalfunction,leadstotheappearanceofmul-Table1.
GeneralbiochemistryinthedifferenttreatmentgroupsSHAM(n=10)CRF8C(n=9)CRF8P(n=9)CRF12C(n=7)CRF12P(n=10)CRF20C(n=10)CRF20P(n=10)Urea(mg/dL)34±9108±27137±45119±29143±51100±70200±70Creatinine(mg/dL)0.
4±0.
11.
0±0.
31.
4±0,51.
3±0.
41.
3±0.
61.
5±1.
32.
2±0.
8*Calcium(mg/dL)11.
4±0.
611.
8±0.
911.
4±0.
812.
4±0.
611.
4±2.
012.
5±0.
611.
1±0.
4*Phosphorus(mg/dL)4.
8±1.
05.
7±1.
110.
0±3.
7*6.
0±1.
99.
4±3.
4*5.
6±0.
812.
1±2.
8*PTH(pg/mL)22(4-74)28(19-55)139(59-933)62(27-121)236(128-1,113)*80(54-115)1.
901(1,117-2,517)*CRF8C,CRF12C,CRF20C:groupsofratsfeddietswithnormalphosphoruscontentsacrificedat8,12and20weeks,respectively;CRF8P,CRF12P,CRF20P:groupsofratsfedahighphosphorusdietsacrificedat8,12and20weeks,respectively;*P2GpolymorphismwithaorticvalvecalcificationCorrespondence:MIsabelRodríguezGarcía-ServiciodeMetabolismoseoyMineral-HospitalUniversitarioCentraldeAsturias-EdificioFINBA-Avda.
deRoma,s/n-33011Oviedo(Spain)e-mail:irodriguez@hca.
esDateofreceipt:29/01/2016Dateofacceptance:06/06/2016WorkawardedascholarshipBasicResearchFEIOMM2013.
SummaryIntroduction:Themostcommoncauseofaorticstenosisisactivecalciumaccumulationinthevalvecusps,whichimpliesseriousclinicalconsequences.
Variousextracellularmatrixmetalloproteases(MMPs)havebeenimplicatedinthedevelopmentofthisdisease.
Therefore,thepossibleassociationbetweenafunctionalMMP1polymorphismandtheamountofcalciumdepositedontheaorticvalveisstudied.
Patientsandmethods:45patientsundergoingvalvereplacementwereincludedinthestudy.
Thecalciumcontentinvalvecuspsremovedduringsurgerywasdeterminedbycomputedmicro-tomography.
DNAwasextractedfromperipheralbloodsamplesforgenotypingthe-16071G>2GpolymorphismofMMP1byPCRandsubsequentdigestion.
Results:Significantdifferenceswereobservedinthecalciumcontentinaorticvalvesinindividualswithdifferent-16071G>2Ggenotypes(p=0.
042).
Thus,2Gallelecarriers(homozygousorheterozygous)pre-senthighercalciumlevelsmeasuredasBMD(p=0.
004)aswellasBV/TV(p=0.
002).
TheassociationwithBV/TVwasindependentofsex,age,degreeofrenalfunctionandanatomyofthevalve(p=0.
02).
BMDtendency(p=0.
07)wasalsoobserved.
Conclusion:Theassociationbetween1G>2GMMP1polymorphismandcalciumcontentoftheaorticvalvesuggeststhatthe1Gallelewouldhaveaprotectiveeffectagainstcalciumdeposits.
Theseresultssupporttheimportanceoffurtherstudytoconfirmwhetherthispolymorphismcouldbeusedasapos-siblepredictorofaorticstenosisdevelopment.
Keywords:aorticvalvedisease,matrixmetalloproteinasepolymorphisms,microCT,calciumcontent.
DOI:http://dx.
doi.
org/10.
4321/S1889-836X2016000400003ORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):115-120116IntroductionAorticstenosisdegenerationisthemostcommonvalvediseaseintheindustrializedcountries1.
Initiallyconsideredapassiveprocess,itisnowdescribedasanactivecalciumbuildupinthevalvecusps,accompaniedbychangesinthemor-phologyandfunctionofvalvularcells,characteri-zedbynotableosteoblastdifferentiationthatincreasesvalvestiffness.
Thisleadstoareductionintheorificeopeningofthevalveandanincrea-seinbloodpressuregradient,withseriousclinicalconsequences2.
Thereisabundantevidencethatimplicatesextracellularmatrixmetalloproteinases(MMPs)inthisprocess3.
MMPsarealargefamilyofzinc-dependentenzymesthatexerttheirfunctioninbothpathologicandphysiologicalconditions4.
Traditionallytheyhavebeengroupedaccordingtotheirabilitytodegradevariouscomponentsoftheextracellularmatrix,butalsoexertfunctionsinotherlocations.
Infact,recentlyresearchershavesuggestedthattheyalsoactonnon-matrixpro-teinsandhighlighttheirroleininflammatorypro-cesses5,6.
Ithasalsobeenfoundthatthereisincre-asedexpressionofMMP-1,-2and-3incalcifiedaorticvalvesthaninnormalaorticvalves,andtheexclusivepresenceofMMP-9indiseasedvalves7.
TheassociationofMMPswithvalvediseasehasalsobeenstudiedfromthegeneticpointofview.
Thus,theMMP11G>2Gpolymorphismgeneat-1607isreportedlyassociatedwiththepresen-ceofbicuspidaorticvalveanamoly8.
Thissamepolymorphismhasalsobeenassociatedwithlevelsofbonemineraldensityinpostmenopausalwomen9.
Basedonthedatapresentedandinordertoidentifyapossibleearlymarkerofdiseasecal-cificaorticvalve,theassociationof1G>2Gpoly-morphismwithparametersindicativeofminerali-zationinaorticvalvesfromvalvereplacementwasstudied.
PatientsandmethodsPopulationgroupAorticvalvesfrom45patientsdiagnosedwithaor-ticvalvedisease(stenosis91%,failure9%)under-goingaorticvalvereplacementbetweenApril2012andMay2014intheDepartmentofCardiacSurgeryoftheCentralUniversityHospitalofAsturiaswerestudied.
Table1showssomefeatu-resofgreaterclinicalinterestareshown,includingcardiovascularriskfactors.
Acurrentsmokerwasconsideredactiveifthatpersonhadsmokeduringthepreviousyear.
Anex-smokerwasanindividualwhogaveupsmo-kingmorethanayearpriorandnon-smokingtoapersonwhohasnevertakenupthehabit.
Dyslipidemiawasdefinedaccordingtocomplian-cewithanyofthefollowingcriteria:historyofdiagnosedhyperlipidemiaand/ortreatedwithmedication,dietand/orexercise,figuresoftotalcholesterolabove200mg/dl,LDLcholesterolgre-aterthanorequalto130mg/dl,HDLcholesterollessthan40mg/dlorlipid-loweringtherapy.
Hypertensionwasdefinedasmeetingoneofthefollowingcriteria:historyofdiagnosedortreatedwithmedication,dietand/orexercisehyperten-sion;systolicbloodpressurelessthan140mmHgordiastoliclessthan90mmHg,atleasttwodeter-minations;orantihypertensivetreatmentnotadmi-nisteredastherapytoanythingotherthanhyper-tensiondisorder.
Theexistenceofdiabetesmelli-tuswasbasedonthepresenceofanyofthefollo-wing:accreditedmedicalhistoryofdiabetesmelli-tus,bloodglucosegreaterthanorequalto200mg/dlfastinginanysituationandsymptomsofdiabetesmellitus,theleasttwodeterminationsofbloodglucosegreaterthanorequaltofasting126mg/dl(fastedunderstoodasaperiodwithoutinta-keforatleast8hours)oruseoforalhypoglyce-miccurrenttreatmentsand/orinsulin.
Theestima-tionofglomerularfiltrationwascarriedoutusingtheMDRD-4variableequation.
Theclassificationofthevalveanatomywasmadebasedonintrao-perativefindings,inadditiontotheECGdescrip-tionpriortosurgery.
Tissuesremovedduringsurgeryweretreatedfor24hwith4%formaldehydeandafterseveralwashingswithwater,preservedin70%ethanolat4°CinthePrincipalityofAsturiasBiobank.
PeripheralbloodsampletubewithEDTA,whichwasprocessedinthebiobankforextractionofgenomicDNAwasstoredat-20°Cuntiluse.
PatientssignedaninformedconsentformforuseoftheirbiologicalsamplesandthestudywasapprovedbytheEthicsCommitteeofthePrincipalityofAsturiasforClinicalResearch.
GenotypingTheMMP11G>2Gpolymorphismatposition-1607(rs1799750)wasgenotypedbypolymerasechainreactionandsubsequentdigestionwithrestrictionenzyme(PCR-RFLP),followingapreviouslyrepor-tedprocedure10.
QuantificationofcalciumcontentintheaorticcuspValvulartissuesamplespreservedinethanolwereanalyzedbycomputerizedmicrotomography(microCT)inaSkyScan1174(Bruker,Kontich,Belgium)availableattheUniversityofOviedoVivariumresearchcenter.
Imageswereobtainedusing50kVand800Aparameters.
1,300imagesofeachofthesampleswithapitchof0.
3°rotationandanaverageframe2fora180°scanwereobtai-ned.
Scanningeachlasted10to20minutes(dependingonvalvesize)usinganexposuretimeof6,200ms.
flatfieldcorrectionatthebeginningofeachscan.
TheimagesobtainedwerereconstructedwiththeNRecon(Bruker)software(Figure1).
Correctionvaluesofattenuationcoefficient,lightraysharpness,smoothnessandringartifactswerethesameinallsamples.
3Dmorphometricanaly-siswascarriedoutusingCTAn(Bruker)software.
Thevolumeofinterestwasmanuallydelimitedineachofthesamples.
Thethresholdusedforallof0.
74to3.
39wasg/cm3ofbonemineraldensity(BMD).
ORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):115-120117BMDparametersandbonevolume/totalvolume(BV/TV)wereconsideredasmeasuresoftheamountofcalciumdeposited.
StatisticalanalysisAllstatisticalanalyzeswerecarriedoutusingSPSSver-sion15.
0software.
ItwasfirstconfirmedthatgenotypeandallelefrequenciesofpolymorphismwereinHardy-WeinbergEquilibriumModelbyχ2test.
ANOVAtestwasusedtocomparethemeanvaluesoftheparame-tersstudiedinthedifferentgenotypesandthenBonferronitesttodiscrimi-natewhichgenotypepairsshowedstatisticalsignifican-ce.
Then,basedontheseresults,thegenotypesweregroupedintotwocategorieswhoseaveragevaluesforBMDandBV/TVwerecomparedbyT.
Finallytest,anadjustedlinearregressionanalysiswasperformedforvariablesofsex,age,glomerularfil-trationratemeasuredusingtheMDRD-4andpre-senceofbicuspidaorticvalveanamoly.
Ap-value2G,polymorphismatposition-1607withafrequencyof0.
49fortheminoralle-le(1Ginourpopulation),similartothatofotherEuropeanpopulations(dbSNP).
TheaveragevaluesofBMDandBV/TVinpopulationgroupsdefinedbydifferentMMP1polymorphismgenotypesatposition-1607anddifferencesfoundinbothvariableswerecalcula-ted,althoughtheywerestatisticallysignificantonlyinthecaseofBV/TV(Table2).
Posthocanalysisfoundthathomozygousindi-vidualspresentedsignificantdifferencesforthe2Gallelecomparedwithhomozygousfor1G(p=0.
042),withcalciumcontentvaluessimilartothoseofhomozygousfor2Galleleheterozygousindividuals.
Thus,applyingamodelofrecessiveforthealleleeffect1G,itwasfoundthatallelecarriers2Ghadsignificantlyhighervaluesofcal-ciumcontentintheaorticvalve(3timesinBMDand2timestheBV/TV)thannoncarriers(BMDvaluesof62.
52±10.
99mg/cm3in2Gallelecarriers±8.
54versus20.
08mg/cm3inthe1Gallelehomozygotes,andvaluesBV/TV5.
44±0.
62%in2Gallelecarriersversus2.
52±0.
59%inhomozygo-tesforthe1Gallele)(Figure2).
ThesesignificantdifferencesremainedforlevelsofBV/TVafteradjustmentforsex,age,presenceofbicuspidaor-ticvalveandglomerularfiltrationrate(p=0.
021),maintainingthetrend,butwithoutbeingsignifi-cant,forBMDlevels(p=0.
073).
DiscussionThisstudyisthefirsttodescribeanassociationbetweenapolymorphismofMMP1geneandtheamountofcalciumintheaorticvalves.
Thelitera-turecontainsvariousassociationsofvariantsofthisgenewithothercardiovascularconditions11-13.
Amongthesevariants,oneofthemoststudiedisthe1G>2Gpolymorphisminthepromoterregionofthegene,fortheinsertionalleleconfersgreatertranscriptionalactivity14whichcanhaveeffectsonthecellandthereforeinthebody.
Thus,asignifi-cantlyincreasedriskofatherosclerosisinthecaro-tidarteryinindividualscarryingthe2Gallele15andagreaterpresenceofthisalleleinpatientswhohadsufferedischemicstrokewasobserved16.
However,untilnowassociationsofthispolymor-phismandcalcificaorticvalvediseasehadnotbeendescribed,althoughtheMMPshavebeenknowntoplayanimportantroleintheirphysio-pathology17,18.
Twoimportantaspectsinthedevelopmentofdiseasearecalcifiedaorticvalveinflammationandextracellularmatrixremodeling5.
Botharemodula-tedbyvalveinterstitialcells(VIC),whichpassfromareststateinwhichtheyretaintissuehome-ostasis,toanactivatedstatewhichtakemyofibro-blasts19.
ActivatedVICrespondtoinflammationbysecreting,amongotherfactors,MMPs,whichwillcontributetotheaccumulationofdisorganizedfibroustissue,tokeepthevalvesinastateofchro-nicinflammationandinduceosteoblasticdifferen-tiationofVIC.
Thelattereventwouldpromoteandacceleratecalciumdeposition,whichwouldresultinreducedfunctionofthevalve20.
Theinteractionbetweencellsandtheextrace-llularmatrixthatcontainsthemisessentialforthephysiologyandfunctionalityofthevalvetissueandaffectstheVICphenotype3.
Theextracellularmatrixoftheheartvalvesismadeupto90%collagenand,infact,excessivedepositionofprotein,accompa-Table1.
ClinicalandanthropometriccharacteristicsofthestudypopulationCharacteristicsValuesAgea(years)69±11Mens63%Smoking17.
4%Dyslipidemia43.
5%Hypertension67.
4%Diabetes21.
7%MDRD-4b(ml/min)82±28Bicuspidaorticvalve20%aaverage±standarddeviation.
bglomerularfiltrationrateinml/min/1.
73m2(mean±standarddeviation).
ORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):115-120118niedbyanalteredalignmentoffibersincreasestis-suestiffness21.
Severalstudieshaveshownthecru-cialroleofcollagenincalcificationofaorticvalves22-24.
Specifically,invitrocultureofporcineaorticvalvecuspsofcollagenasetreatedwithincreasedcollagenandsimultaneouslyadecreaseintheamountofothercomponentsoftheextracellularmatrixsuchashyaluronicacidhasbeenobserved23.
Also,anincreasewasdetectedinbothproliferationandapoptosisofVIC,whichexpressedmarkersassociatedwithamyofibroblastphenotype(alpha-smoothmuscleactin)andosteoblast(alkalinephosphatase,osteocalcinandbonesialoprotein)resultinginincreasedtissuemineralization23.
MMP-1,alsoknownasfibroblastcollagenase,degradesinterstitialcollagentypesI,IIandIII.
Consequently,itsincreasedactivitywouldpromo-tethedestructionofcollagen,osteoblastdifferen-tiationandcalcification.
Theresultsobtainedinthisstudysupportthisreasoning,associatingthehighestamountsofcalciuminvalveswiththoseindividualscarryingthealleleresultinginincrea-sedtranscriptionofthegeneand,consequently,agreateramountofMMP-1protein.
Viewedanotherway,thelackoftheprotectiveeffectbroughtaboutinpatientslesstranscript(providedbythelessactiveallele)facilitatingthedevelopmentofcalcificationintheaorticvalve.
Alimitationofourstudyisthesmallnumberofpatientsincludedandthefactthatitisacross-sec-tionalstudy.
However,anassociationwith1G>2Gpolymorphismhasbeenaffirmed,suggestingthepro-tectiveeffectofthe1Gallelewillnecessitatestudyinglar-gersamplesandotherpopu-lationgroupsinordertoascertainwhetherthisfin-dingcouldbeusedinthefutureasapredictorofcalci-ficationandaorticstenosis.
Competinginterests:Theauthorsdeclarethattheyhavenoconflictofinterest.
Funding:ThisworkwassupportedinpartbytheprojectoftheInstituteofHealthCarlosIIIPI10/00173-FEDER.
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Figure1.
Imagesobtainedafteranalysisbycomputerizedmicro-tomography(microCT)oftheaorticvalvecusps.
4Examplesofvalvecuspsareshownwithvaryingdegreesofcalcification119ORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):115-120Table2.
ValuesofBMDandBV/TVforeachgenotypepolymorphismofMMP1atthe-1607positionFigure2.
ValuesofBMDandBV/TVforMMP1-1607genotypesasamodelofrecessiveinheritanceforthe1Gallele.
Average±standarderrorisshown.
*p=0.
004;#p=0.
002Genotype-1607MMP1BMD(mg/cm3)BV/TV(%)2G2G(N=13)66.
72±65.
315.
87±3.
761G2G(N=20)59.
79±63.
225.
16±3.
511G1G(N=12)20.
08±29.
592.
52±2.
03Total(N=45)51.
20±58.
964.
66±3.
46p-valor0.
0950.
033Thedataarerepresentedasmean±standarddeviation.
P-valueobtainedbyANOVA.
*BMDBMD(mg/cm3)807060504030201002G2G+1G2G1G1GGenotype-1607MMP1#BV/TVBV/TV(%)765432102G2G+1G2G1G1GGenotype-1607MMP113.
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Haplotypeeffectofthematrixmeta-lloproteinase-1geneonriskofmyocardialinfarction.
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Matrixmetalloproteinase-1andmatrixmetalloproteinase-12genepolymorphismsandtheriskofischemicstrokeinaTunisianpopulation.
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2016;8(4):121-126121Pérez-CampoFM2,SaudoC1,KrebesovaR1,Delgado-CalleJ3,RianchoJA11DepartamentodeMedicinaInterna-HospitalU.
M.
Valdecilla-UniversidaddeCantabria-IDIVAL-Santander(Espaa)2DepartamentodeBiologíaMolecular-UniversidaddeCantabria-Santander(Espaa)3DepartamentodeAnatomíayBiologíaCelular-FacultaddeMedicinadelaUniversidaddeIndiana-CentroMédicodeAdministracióndeVeteranosRoudebush-Indianápolis-Indiana(EE.
UU.
)FunctionalstudyofpromotergenepolymorphismsofsclerostinCorrespondence:JoséA.
Riancho-DepartamentodeMedicinaInterna-HospitalU.
M.
Valdecilla-Avda.
Valdecilla,sn-39008Santander(Spain)e-mail:rianchoj@unican.
esDaeofreceipt:16/07/2016Dateofacceptance:25/09/2016WorkrewardedwiththescholarshipofMolecularBoneBiologyFEIOMM2013.
SummarySclerostin,encodedbytheSOSTgene,inhibitstheWntpathwayand,consequently,tendstodecreasebonemass.
SomepolymorphismsoftheSOSTpromoterhavebeenassociatedwithbonemineraldensity(BMD),butthemolecularmechanismsinvolvedareunknown.
Theaimofthisstudywastostudythefunctionalroleofonepolymorphisminvitro.
WeclonedtheproximalpromoterregionofSOSTgene,containingdifferentallelesatthers851054SNP,inluciferasereportervectorsandtransfectedthemintothecelllinesHEK-293T,SAOS-2andHOS-TE85.
Wedidnotfindsignificantdifferencesinthetranscrip-tionalactivityofvectorswitheithertheAortheGalleleoftheSNP.
Theco-transfectionofvectorsexpres-singRUNX2andOSXmarkedlyincreasedthetranscriptionalactivityoftheSOSTpromoterconstructs(Aallele,2.
5±0.
9fold,p30min/dayBreastcancer915Prevalenceincases0.
63Controls1537Prevalenceincontrols0.
71Chi-squared=0.
24p=0.
625Oddsratio1.
48CI95%0.
53-4.
11Populationattributablerisk0.
12Attributableriskinexposed0.
81DeficientvitaminDintakeDeficientSufficientBreastcancer186Prevalenceincases0.
75Controls2230Prevalenceincontrols0.
42Chi-squared=5.
79p=0.
016Oddsratio4.
09CI95%1.
04-11.
9Populationattributablerisk2.
81Attributableriskinexposed3.
33Overweight/obesityBMI>25BMI18-24.
9Breastcancer186Prevalenceincases0.
75Controls2131Prevalenceincontrols0.
59Chi-squared=6.
55p=0.
01Oddsratio4.
42CI95%1.
51-13.
1Populationattributablerisk3.
09Attributableriskinexposed4.
12VerylowlevelsofvitaminD≤20ng/mL>20ng/mLBreastcancer186Prevalenceincases0.
75Controls1339Prevalenceincontrols0.
25Chi-squared=14.
99p=<0.
001Oddsratio9.
0CI95%2.
95-27.
5Populationattributablerisk6.
64Attributableriskinexposed0.
88BMI:bodymassindex;95%CI:confidenceintervalof95%.
132ORIGINALS/RevOsteoporosMetabMiner.
2016;8(4):127-133bonehealthandprotectionagainstotherchronicdiseasesassociatedwithadequatevitaminDlevelsareequallyimportant.
WemustencouragepatientstopracticehealthylifestylehabitsthathelpincreaselevelsofvitaminD,suchasmaintai-ninghealthyweight,avoidsmoking,increasephysicalactivity,andappreciatetheimportanceofvitaminDsupplementsinadultswhodonotobservethesechangesinlifestyle.
Competinginterests:Theauthorsdeclarenoconflictsofinterest.
Acknowledgments:Theauthorsofthismanus-criptthankBoehringerIngelheimMexicoforitssupportforstatisticalanalysisandfinalediting.
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134CLINICALNOTE/RevOsteoporosMetabMiner.
2016;8(4):134-137MontaoJaramilloD,DíazCurielMUnidaddeEnfermedadesMetabólicasseas-ServiciodeMedicinaInterna-FundaciónJiménezDíaz/QuirónSaludMadrid-Madrid(Espaa)Hemochromatosisandosteoporosis,inreferenceto4casesCorrespondence:ManuelDíazCuriel-FundaciónJiménezDíaz-Avda.
ReyesCatólicos,2-28040Madrid(Spain)e-mail:mdcuriel@fjd.
esDateofreceipt:05/09/2016Dateofacceptance:12/12/2016SummaryAlthoughmostpeople'sosteoporoticconditionstreatedinclinicalpracticemaybecategorizedinthepostme-nopausalosteoporosisgrouporrelatedtoaging,therearesomeosteoporosiscaseslinkedtothedevelopmentofsomeotherdiseaseoridentifiablefactor.
Mostofthesecausesareassociatedwiththetakingofsteroids,hypogonadism,malignantprocessessuchasmultiplemyeloma,gastricsurgery,alcoholismandtreatmentwithanticonvulsantdrugs.
Hereditaryhemochromatosisisanotherdisorderrelatedtotheonsetofosteoporosis.
Inthispaper,wepresent4casesofpatientswithosteoporosiswhoalsosufferhereditaryhemochromatosis.
Thelatter'scharacteris-ticsaredescribedandalsoitspossiblerelationshipwithbonedisease.
Keywords:hemochromatosis,osteoporosis,iron.
DOI:http://dx.
doi.
org/10.
4321/S1889-836X2016000400006135CLINICALNOTE/RevOsteoporosMetabMiner.
2016;8(4):134-137IntroductionOsteoporosis(OP)isadiseasecharacterizedbydecreasedboneresistancewithbonemassqualityandquantityalterationthatleadstodiseasefractu-resorfragility.
Theforearm,thevertebralbodiesandthehiparethemostcommonlocations.
Riskfactorsincludeage,earlymenopause(andanycaseoflowestrogenproduction),fracturesduetopreviouspersonalfragility(thisbeingthemostimportantriskfactor)2orinfirst-degreerela-tives,inadequateintakeofdairyproducts,chronicgluco-corticoidintake(prednisoneatdoses≥5mg/dfor3months),lowbodymassindex(BMI)(<19mg/m2),highandchronicalcoholandcaffei-ne,andsmoking,aswellasallthosediseasesthatmaycauseasecondaryOP(hypogonadism,hyperthyroidism,diabetesmellitus,renalfailureandliverdiseases,amongothers).
Hereditaryhemochromatosis(HH),althoughuncommon,isoneoftheliverdiseasesdescribedthatcanleadtotheonsetofOP.
Themostfre-quentformofpresentationisrelatedtotheHFEgene(HH-HFE).
HerewedescribetheexistenceofOPin4peoplesufferingfromHH-HFE.
ClinicalCasesAtourhospital'smetabolicbonediseasecenter,4womenwerediagnosedwithOPandassufferingfromHH.
WedonotknowtheactualincidenceofOPinpatientswithHHinourcenterasboneden-sityDXAhasnotyetbeencarriedoutonallofthesepatients.
1stpatient:ShewasdiagnosedwithHH-HFEandpituitaryhypogonadismat25yearsandwithOPat51years.
Giventhehistoryofhypogonadism,hor-monereplacementtherapywasstartedforamenor-rheaandsubsequentlymodifiedtoraloxifenewithsupplementsofcalciumandvitaminD,withperio-dicfollow-up.
Inthelastreview,DXApresentedaT-scoreof-2.
1inthefemoralneck(stableduringtre-atment)and-3.
2inthelumbarspine(withslightdeteriorationsinceonset,sincewestartedfrom-2,8).
Shedidnotpresentfracturesduringthistime.
WhenstudyingtheriskfactorsofOP,itwasobservedthatthepatientpresentsadequatecalciumintake,exerci-sesregularly,isanon-smokerandhasanadequatethyroidfunction.
HH-HFEdidnotcauseorganicinvolvement,sinceithadbeenfollowedandcontro-lledsinceitsdiagnosis,and2phlebotomieswereperformedduringfollow-up,withnormalizationoftheanalyticalparametersofiron;shehasalwayspre-sentedalkalinephosphatasewithinnormallimits.
2ndpatient:ShewasdiagnosedwithOPandHH-HFE(normalheterozygote/H63D)atage64,althoughshehadahistoryofvertebralfracturesatage55andribsatage61and63years.
Shehasbeentreatedfor4yearsinourcenter,andthelastDXAdetectedaT-scoreinthefemoralneckof-2.
6(stableduringfollow-up)andinthelumbarspineof-2.
1(slightimprovementwithrespecttoinitia-tionoftreatment,-2.
7).
InitiallyshewastreatedwithcalciumandvitaminD.
Subsequently,iban-dronicacidwasaddedandthiswaslaterreplacedbydenosumabuntilthepresenttime,asthepatienthasnotpresentedanynewfractures.
AsforOPriskfactors,shepresentslowcalciumintakeinthediet,isasmokerof40cigarettes/dayandhadmenopau-seat45years.
HH-HFEdidnotproduceorganicinvolvementsinceitwasdiagnosedandatnotimediditrequirephlebotomies.
Inherfollowupshehasalwayspresentedalkalinephosphataseinthenormalrange.
Asasignificantpersonalhistory,at66yearsthepatientwasdiagnosedandsurgicallytreatedforcoloncarcinomaand,inaddition,isaheterozygouscarrieroftheprothrombinmutation.
3rdpatient:OPwasdiagnosedat69yearsofageandat74yearsofHH-HFE(heterozygoteforC282Y)duetoalterationsintheferricprofile,whichwerealreadyobservedatthetimeofPBdiagnosis.
ShewastreatedwithcalciumandvitaminDduringthe5yearfollow-up,andinthelastDXAshepre-sentedaT-scoreatthefemoralneckof-1.
1(wor-seningwithrespecttothetimeofdiagnosis:0.
3,butslightimprovementcomparedtothepreviousone:-1.
5)and-2.
3inthelumbarspine(slightimprovementcomparedtothestart:-2.
6).
Hedidnotsufferfracturesduringtheseyears.
AsforherriskfactorsforOP,shehas2childrenwithwhomshebreastfeedandpresentedmenopauseatage45,isanex-smoker,hasalowintakeoffoodsrichincalcium,doesnotpracticephysicalexerciserouti-nelyanddoesnothaveahistoryoffractures.
HH-HFEisadequatelycontrolledwithoutorganicalte-rationsandatnotimeneededphlebotomies;Duringhisfollow-uphealwayspresentedalkalinephosphataseinthenormalrange.
Asanimportantpathologicalantecedent,hewasdiagnosedofchro-nichepatitisCvirusinresponsetoantiviraltreat-ment,whichwasmaintained9yearslater.
4thpatient:DiagnosedHH-HFEandlaterlumbarOPat55years.
Ithasbeenfollowedinourcon-sultationfor8years,andinthelastDXApresen-tedaT-scoreatthefemoralneckof-1.
1(beingtheinitialof-1.
6)andinthespineof-2.
8(maintainedstablewithrespecttothestartoftreatment).
Atthebeginningandduringherevolutionshewastrea-tedwithcalcium,vitaminDandraloxifene,withouthavingpresentedfractures.
AsforherOPriskfactors,shehas1childanddidnotbreastfe-ed,hadmenopauseatage51,isnotasmoker,hasadequateintakeofcalciuminthedietandexerci-sesroutinely.
Shehasnoprevioushistoryoffrac-ture.
ShedidnotpresentorganicalterationsbyHH-HFEanditwasnotnecessarytopracticephle-botomiesduringherfollow-up.
Thealkalinephos-phatasewasalwayswithinthenormalrange.
Asanimportantpersonalantecedent,shewastreatedforchronichepatitisC,withadequateresponse.
DiscussionHepaticosteodystrophyreferstoosseousdiseases(mainlyOPandosteomalacia,althoughthelatterisveryrare)secondarytochronicliverdisea-ses1,2,6,12,suchasHH-HFE.
CLINICALNOTE/RevOsteoporosMetabMiner.
2016;8(4):134-137136HHisadiseasecharacterizedbyincreasedtis-suedepositsofironsecondarytodecreasedpro-ductionorresistancetohepcidin,ahormonethatinsituationsofexcessirondecreasestheintestinalabsorptionofironbytheenterocytesandtherele-aseofironbymacrophages1,10.
Thisraisesbloodironanditsreservesintheorganismwiththecon-sequentformationofpathologicaldepositsinvarioustissues(liver,heart,pancreas,joints,bones,pituitaryandskin,amongothers),genera-tingmultiplesymptomsdependingontheirloca-tion.
Mostpatientsareasymptomaticintheearlystagesofthediseaseastheydonotyethavethesedeposits.
ItshouldbenotedthatpatientswithHHpresentanincreasedincidenceofcirrhosisandhepatocellularcarcinoma1,3,11.
HHisapredominantdiseaseintheCaucasianpopulationand5typeshavebeendescribed.
ThemostcommonisinheritedgeneticalterationinanautosomalrecessiveformrelatedtotheHFEgene,themostseverepresentationbeinghomozygousC282Y,andtheleastrelevantH63D10.
ItshouldbenotedthatthefactthatapatientishomozygousfortheC282YmutationisnotadiagnosisofHHifthereisnoelevationoftheironstores1,sincetheHFEgenehasquiteavariablepenetranceanditisnotpossibletoascertainwhichpatientshomozy-gousforC282Ywilldevelopthedisease3.
Amongtheconditionsrelatedtoironoverload,HH-HFEisthemostcommonandtheoneinwhichmostsignificantclinicalcomplicationsoccur,althoughwealsofindlesscommonsecon-darycauses,suchasthalassemiamajor,sideroblas-ticanemia,multipletransfusions,long-termhemo-dialysis,chronichepatitisBandC,alcoholicandnon-alcoholicliverdisease,amongothers.
Thetreatmentforironoverloadinvolvesperio-dicphlebotomiesuntilthenormalizationoftheanalyticalparameters,whichnotonlymanagetocontrolthelevelsofirondepositsinthebody,butalsoimproveandsometimessecondarycomplica-tionsofthisdiseasedisappear1.
Asforbonetissue,arthritisandOPhavebeenfoundtobethemainbonealterationsrelatedtoHH-HFE,witharthritisbeingthemostcommonlyassociated,reachingupto80%ofpatients.
Treatmentwithphlebotomiesdoesnotmanagetocompletelyreversethisonceitisalreadyestablis-hed1,9.
TheassociationbetweenOPandHHhasbeenknownsince1960andtheincidenceofOPisapproximately25-45%5-7,9.
InastudyconductedinBrazil,thepresenceofarthropathy,hepatoce-llularcarcinoma,osteoporosisanddiabeteswasmorecommoninHH-HFEpatientscomparedtopatientswithironoverloadfromothercauses3.
ThemechanismsbywhichOPoccursinchro-nicliverdiseasesarenotfullyknown,butinHH-HFEitisthoughtthatincreasedbloodiron,notcirrhosis,isthemaincauseofthisassociation,althoughincirrhoticpatients,involvingahigherincidenceofhypogonadism,itisknownthatthenumberofOPcasesincreasescomparedtopatientswithouthypogonadismorcirrhosis2,5,7,9.
AdvancedHHmayleadtocirrhosis(withorwithouthypogonadism)whichaddstothedelete-riouseffectofexcessirononthebone.
In1989,TerrenceDiamondetal.
consideredthatexcessserumironalteredthefunctionofoste-oblastsbydecreasingosteoidmatrixsynthesis7,whichhasbeencorroboratedbytwootherstu-dies,bothinvitro,oneofwhichconcludesthatelevationofserumirondecreasesboneminerali-zationWithincreasingferritinanditsferoxidaseactivity,sinceitaltersthefunctionofosteoblastsbymodifyingtheactivityofthegenesofCBF-α1(involvedinthematurationanddifferentiationofosteoblasts),osteocalcinandalkalinephosphataseindose-dependentform4.
TheotherstudyalsoconcludedthatironoverloadproducesOPbyinhibitingtheproliferation,differentiationandmineralizationofosteoblasts,aswellasdecreasedalkalinephosphataseactivity8.
ThishasalsobeenstudiedbyValentietal.
whoconsiderthatOPinHHisrelatedtohypogona-dism,severityofironoverloadandlowweight,differinginalkalinephosphatase,sincetheyfoundthathighlevelsarealsocorrelatedwithOP.
Thismaybeduetoitsrelationtohypogonadism(inwhichboneresorptionisincreased,sinceitstimu-latesosteoclastactivity)5,7,12.
AsuitableDXAdiagnosisshouldbeperformedforallpatientswithchronicliverdisease,inclu-dingHH(especiallyHH-HFE)2,5,12,althoughthetimeframedeterminationforperformanceofthisstudyinthefollow-upofpatientshasnotbeendetermined2,6.
ThemostcommonlocationofT-scoredecreaseinDXAinpatientswithHH-HFEisthelumbarspine,followedbythefemoralneck5,9.
RegardingthetreatmentofOP,followingthesameguidelinesisrecommendedasinpatientswithouthepaticdisease,adjustedaccordingtoindividualcharacteristics.
Avoidinghormonereplacementtherapyinsevereliverdiseaseisalsorecommended12.
ToreduceironoverloadinHH-HFEpatients,periodicphlebotomieshavebeenshowntoimprovetheferricprofileandthusimproveosteoblasticfunction,whichcansometi-mesbereflectedinadecreaseintheT-scorevalueoftheDXA5,7,12.
Asforthe4patientspresentedhere,HH-HFEwasdiagnosedbasedonanalyticalalterationsatanearlystage,andwithoutanytargetorgandama-geinanyofthecases.
TakingintoaccountthatthisdiagnosishasprecededorbeenperformedsimultaneouslywiththatoftheOP(exceptforthe3rdpatient,althoughtheanalyticalalterationswerealreadypresentatthetimeofOPdiagnosis),wemayconsideritariskfactorassociatedwitheachpatient'sotherclinicaldataandnotasthemaincauseofOP,sothatmonitoringandanalyticalcontrolhavebeenperformedtotreatitandavoidcomplicationsofHH-HFEaswouldbedoneinanypatientwithoutOP.
Inthefirstpatient,hypogonadismisalsoanasso-ciatedriskfactorforpresentingOP,althoughthispathologyhasalwaysbeencontrolledbythegyne-cologyservicesinceitsdiagnosis.
Thenextpatienthaslowcalciumintakeandisasmoker,whichalsoCLINICALNOTE/RevOsteoporosMetabMiner.
2016;8(4):134-137137contributestothepresenceofOPtogetherwithHH-HFE.
Beingasmoker,lowintakeofcalcium-richfoodsandlackofphysicalexerciseareotherriskfac-torsassociatedwithOPthatthethirdpatientpre-sents.
Shealsopresentedslightalterationsoftheferricprofilewithoutneedingphlebotomiestocorrectit.
Finally,thelastpatienthadnootherasso-ciatedfactorsexcepttreatedhepatitisC,althoughshedidnotdevelopcirrhosisorothercomplications.
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2016;8(4):138-146138Portal-NúezS1,4,delaFuenteM2,4,DíezA3,4,EsbritP1,41readeReumatologíayMetabolismoseo-InstitutodeInvestigaciónSanitaria-FundaciónJiménezDíaz-UAM-Madrid(Espaa)2DepartamentodeFisiologíaAnimalII-UniversidadComplutense-Madrid(Espaa)3HospitaldelMar-IMIM-UniversidadAutónomadeBarcelona-Barcelona(Espaa)4RedTemáticadeInvestigaciónCooperativaenEnvejecimientoyFragilidad(RETICEF)-InstitutodeSaludCarlosIII-Madrid(Espaa)OxidativestressasapossibletherapeutictargetforosteoporosisassociatedwithagingCorrespondence:SergioPortalNúez-readeReumatologíayMetabolismoseo-InstitutodeInvestigaciónSanitaria-FundaciónJiménezDíaz-Avda.
ReyesCatólicos,2-28040Madrid(Spain)e-mail:sportal@fjd.
esDateofreceipt:19/02/2016Dateofacceptance:13/06/2016SummarySenileorinvolutionalosteoporosisisamajorprobleminthedevelopedworld.
Recentstudiespointtoincreasedoxidativestressassociatedwithaging,whetherbiologicalorchronological,asanimportantfac-torinitsdevelopment.
Inthisreviewpaper,wefocusonbonetissuedisordersrelatedtoaging,thesour-ceofoxidativestressandnegativeinfluenceonbonetissue.
Finally,weconsiderthepotentialoxidativestresstherapiescurrentlybeingdevelopedforthisdisease.
Keywords:oxidativestress,osteoporosis,aging,fragility.
DOI:http://dx.
doi.
org/10.
4321/S1889-836X2016000400007REVIEW/RevOsteoporosMetabMiner.
2016;8(4):138-146139IntroductionTheagingpopulationindevelopednationshasledtoanincreaseintheprevalenceandinciden-ceofosteoporosis.
Anestimated200millionpeo-plesufferwiththisconditionworldwide1.
Definedasadecreaseinbonemassandqua-litythatincreasestheriskoffracture2,osteoporo-sisiscloselyrelatedtoaging.
Althoughthefactorsinvolvedhavenotbeenfullyidentified,thoseassociatedwithinvolutionalosteoporosisincludeestrogenaftermenopause3,glucocorticoiddeficittherapy4,diabetesmellitus(DM),primarilytype25;renalfailure6(whichcausessecondaryhyperpa-rathyroidism)and,morerecently,increasedoxida-tivestressassociatedwithmanyofthesecondi-tions7.
Inthisreviewpaper,weconsidertheroleofoxidativestressinbonemetabolismaswellaspossiblealternativedrugtherapytomitigateharm-fuleffectsincasesofosteoporosis.
BonedisordersassociatedwithagingBonetissueundergoesacontinuousremodelingprocess,withconsiderableregenerativecapacityandadaptationtophysiologicalchanges.
Thispro-cesstakesplaceinso-calledboneremodelingunits,consistingofdifferentcelltypes:osteoclasts,osteoblastsandosteocytes(fullydifferentiatedosteoblastsembeddedinthemineralizedmatrixandactualorchestratorofremodelingprocess)8.
Boneremodelingishighlyregulatedbygenetic,mechanical,hormonalandlocalfactorswhichdeterminetheoutcomeofbonebalance.
Peakbonemassisreachedduringpubertyinwomenandsomewhatlaterinmales.
Thelattergroupdevelopahigherbonemassandpresentlar-ger,widerbones,whilethefemalebonestructuretendtobesmallerindiameterandcorticalthick-ness.
Fromabout30yearsofage,anegativebonebalanceisobservedinbothsexes(withapredomi-nanceofboneresorption)whichleadstoagraduallossofbonemasssimilarinbothsexes,initiallyinthetrabecularboneandlaterinthecortical3.
Thisdeclineisacceleratedaftermenopauseinwomenduetolossofestrogens,agentswithprovenantio-xidantproperties,whichmaintainlowerbonemassthaninthecaseofmenduringaging.
Withage,metabolicdisordersthataffectthebonesoccur:neu-romuscularchangesrelatedtolackofmobility;increasedendogenousglucocorticoidproductionandrenalfailurewithdecreasedsynthesisofcalci-triol.
Moreover,withaging,bonecollagenfibersundergostructuralchangesandthebonelosestheabilitytorepairmicrofractures9.
Allthiscontributestotheincreasedincidenceoffractures.
Mostcurrentconceptsonthedevelopmentofsenileosteoporosishavebeenobtainedfromstu-diesinexperimentalmodels,mainlyinrodents.
However,wheninterpretingtheseresults,somebonepeculiaritiesinrodentscomparedtohumansmustbetakenintoaccount,suchascontinuousmodelingbonefromthegrowthplate,theabsenceofmenopause,aswellasalackofHaversiancorti-calbonesystem.
However,asinhumans,rodentshaveshownbonemasslossandadeteriorationofstructureandoflongboneregenerativecapacityassociatedwithaging10,11.
Thebonelossinagedratsisrelatedtoadecreaseinosteoblastmaturationandtheincreasednumberofosteoclastscomparedtoosteoblastsinthetrabecularbone12.
Also,ininbredmiceinwhichbonemassisregulatedprimarilybygeneticfactors,bonelossassociatedwithagemayassumeupto10%ofthetotalbonemass,whichisattributedtodecreasedboneremodeling13-16.
Asobservedinrodents,humansinitiallytendtolosetrabecularbonewithage,especiallyinwomen17,relatedinparttoadecreaseinphysicalactivityand,therefore,themechanicalstimuliinthetissue18.
From70years,decreasedcorticalthicknessismorepro-nouncedwithaconcomitantincreaseintheintra-corticalporosityofthefemur.
Themedullarareaincreasesbothinmenandwomen19.
Thesechangesareassociatedwithincreasedriskofosteoporoticfractures.
However,inbothmiceandhumans,themechanicalpropertiesofbonearerelativelyconser-vedthroughasustainedincreaseinsub-periostealmineral,whichincreasesinertiatime20.
MechanismsassociatedwithboneagingTheunderlyingmolecularmechanismsofinvolutio-nalosteoporosishavebeguntobeelucidatedinrecentyears.
Associatedwithage,therehasbeenadecreaseintheosteoprotegerin(OPG)ratio/ligandreceptoractivatorofnuclearfactor(NF).
Thisratioisanimportantmodulatoroftheremodeledbone21.
BothOPGandRANKLareproducedandsecretedintotheextracellularmediumbyosteoblasticcellsandosteocytes.
Infact,studiesinmicemodelsindi-catethatosteocytesproducemostRANKL,thusdirectlyinfluencingboneremodeling22,23.
OPGisasolubledecoyreceptorthatcapturesRANKLintheextracellularmedium(oronthesurfaceofosteo-blasts)andpreventsitfrombindingtoitsreceptor(RANK)incellsofosteoclasticlineage,therebypre-ventingthematurationandactivationofosteoclasts.
Thus,theOPG/RANKLrelationshipisanimportantanabolic/catabolicbalancefactorduringboneremodeling24.
Thus,thedecreasedOPG/RANKLrelationshipwithageisconsistentwithincreasedosteoclastprecursorsinthebonemarrowofoldmice25.
Osteocyteapoptosisplaysanimportantroleinbonelossassociatedwithageandtoimmobili-zationorlackofstimuli26-28andalsoassociatedwithanincreasedRANKLexpression21.
Moreover,inoldmiceoftheC57BL/6strain,anincreaseinthepro-ductionofendogenousglucocorticoidshasbeenobservedthroughtheactivationoftheenzyme11beta-hydroxysteroiddehydrogenasetype1.
Thisisrelatedtoreducedviabilityofbonecells(osteo-blastsandosteoclasts)andangiogenesis,akeypro-cessinboneformation29.
Severalfactorsmayaffecttherateoffracturerepairwithage30.
Withaging,thereisadecreaseinbonemarrowosteoprogenitor,whichoccursinparallelwithincreasedadipognesis31.
Bothosteo-blastsandadipocytesshareamesenchymalpre-cursorcelldifferentiableeitherlineagedependingonthemicroenvironmentwhichareexposedthesecells.
Furthermore,osteoblastsfromoldREVIEW/RevOsteoporosMetabMiner.
2016;8(4):138-146140miceRANKLproductionincreaseparalleltothedecreaseinexpressionofOPG.
Thisalterationresultsinincreasedosteoclastogenesisandosteo-clastactivity21,25.
Itisnoteworthythatthereareadecreasednumberofendothelialcellsandangio-genesis,whichmaycontributenegativelytotheprocessofbonerepairinolderpeople32.
RecentlyanincreaseinbonemassandreducedriskoffractureshavebeenobservedinelderlysubjectswhoundergoangiotensinIIreceptorantagonisttreatment33.
Thedrug'sapparentbene-ficialeffectontheboneisattributedtotheinhibi-toryactionofangiotensinIIonvariousosteoblastdifferentiationmarkers,suchasrunt-relatedtrans-criptionfactor2(Runx2),essentialforosteoblastdifferentiation,osteocalcin34andtheincreaseofRANKL,whichfavorsosteoclastdifferentiation35.
Thesedatasuggestthathighbloodpressurewhichisprevalentintheelderlycouldalsocontributetoinvolutionalosteoporosis.
Sclerostin,theosteocyte-derivedproductoftheSostgene,isapotentinhibitorofboneformationthroughthebindingtoreceptorsassociatedwithlowdensitylipoprotein5and6,inhibitingthecanonicalWnt.
Recentstudieshaveshownthatcirculatingsclerostinincreasesinpost-menopausalwomenandwithageinbothsexes,whichcouldhaveanegativeinfluenceonbonemass36,37.
Currently,theproductoftheklothogeneisknowntobeanimportantmodulatorofcellularaging38,atransmembraneproteinactingasfibroblastgrowthfactor(FGF)co-receptor23producedbytheosteocytesandinducerofphosphaturia.
Micedefi-cientintheKlothogenesufferacceleratedagingandosteopeniacharacterizedbyadecrease(20-40%)ofcorticalthicknessinthefemur,tibiaandvertebrae,andlowboneremodelingwithaverysharpdeclineincorticalboneformation.
Stromalcellsfromthebonemarrowofthesemicehaveareducedabilitymineralizednoduleformationandphosphatasealka-lineactivity39.
Paradoxically,theseKlothodeficientmicehaveincreasedtrabecularboneinthevertebraeandthemetaphysisoflongbones;aneffectwhichtheauthorsattributetoaselectiveactivationoftheWntpathwayonthetrabecularcomponent.
KlothointeractswiththeWntpathwaythroughitssecretedproduct,whichbindstoligandsofthispathwaybyinhibitingitsaction,hencetheabsenceofKlothocouldleadtoactivationofthepathwayWnt39.
Furthermore,micewithouttelomerasehavebeenshowntoexhibitincreasedcellularsenescenceandadecreaseinbonemass3monthsfrombirth,asso-ciatedwithareductioninboneformationandoste-oblastogenesis40.
Apparently,thisreductionisbecau-semicewithouttelomerasehavepoorlydifferentia-tedosteoblastsandthepro-inflammatoryenviron-mentthatpromotesosteoclastactivity.
OxidativestressasapathogenicfactorininvolutionalosteoporosisAgingcanbeseenasaconsequenceoftheimba-lancebetweenoxidizingagentsproducednatu-rallyincellmetabolismandantioxidantdefenses,withapredominanceofthefirst.
Thisisknownasoxidativestress,whichinvolvestheoxidationofbiomoleculesandfunctionallossofcells41,42.
Increasedoxidativestress,carriedoutprimarilyinthemitochondria,isbasedontheoverproductionofreactiveoxygenspecies(ROS)suchassupero-xideanion(O2.
–),hydroxylradicals(OH)andhydrogenperoxide(H2O2).
Thisincreasecannotbeproperlybalancedbyantioxidantssystemssuchassuperoxidedismuta-se(SOD),catalase(CAT)enzymesglutathionecycle(glutathionereductaseandglutathionepero-xidase)andthioredoxin,amongothers.
ExcessROSwithchronological(and/orbiological)ageoxidizesDNA,proteinsandlipidsandinducesthephosphorylationofmitochondriap66shcprotein,leadingtocelldeath7,43-45(Figure1).
Recently,oxi-dativestresshasbeenfoundtohaveimportantfunctionsincellsignaling46,47.
Inthiscontext,ROScanbeconsideredsecondmessengersofinflam-matoryresponse.
Infact,oxidationandinflamma-tionaretwocloselyrelatedprocessesthatincrea-sewithage48.
Althoughsomeresearchershaveraisedques-tionsaboutwhetheroxidativestressisacauseorconsequenceofaging,inrecentyearsithasbeenimplicatedinthebonedeterioration49.
Usingvariousanimalmodels:prematureaging,osteopo-rosisduetoestrogendeficit(afterovariectomy)ordiabetes,increasedoxidativestressmarkerswasfoundtodecreaseboneformationmechanisms50-54.
Theeffectsofoxidativestresstoinducedeleteriouseffectsonbonetissuearenotyetwellknown.
IncreasedROSmaystabilizeforkheadboxO(FoxO)transcription,animportantfamilyoftrans-criptionregulatorsofmanygenes.
Itsfunctionsincludecontrolofglucosemetabolism,tumorigene-sisandcelldefenseagainstoxidativestress55.
FoxO1and3areexpressedinthebone56,wheretheyseemtoplayakeyroleinmaintainingboneforma-tion56.
IthasbeenshownthatgeneticdeletionofFoxOsinmiceincreasesoxidativestressinboneandinducesbonelosstrabecularandcortical,asso-ciatedwithincreasedosteoblast/osteocyticapopto-sisandadecreaseboneformation57.
TheactivationinvolvesFoxOphosphorylationengagementwiththebeta-catenin57causinggeneinductionofoxida-tivestressresponse,asGADD45andCAT58.
Infact,theprotectiveactionofoxidativestressofKlothoproteinappearsaforementionedmediatedactiva-tionFoxOs39.
Furthermore,activationoftheFoxOpreventsbeta-catenintoactastranscriptionfactorinstimulatingtheproliferationanddifferentiationofosteoblasts56.
IncreasedROSinbonecellscausesdamageandapoptosisgenomicDNAofosteoblastsandosteocytes.
Inaddition,lipidperoxidationdepen-dentlipoxygenaseactivatedbyoxidativestressplaysanimportantroleinbonelossassociatedwithaging.
ThisisevidencedbyanalyzingtheexpressionofthelipoxygenaseandALOX12andformationAlox154-hydroxynonenal,aproductoflipidperoxidation,increasedboneinoldermice59.
Ithasalsobeenshownthatproductsoflipidoxi-dationinhibitingactionosteogenicfactors60.
REVIEW/RevOsteoporosMetabMiner.
2016;8(4):138-146141Furthermore,theincreaseofROShasbeenlin-kedtoanincreaseofosteoclastogenesisandoste-oclastactivity61,62.
Ithasrecentlybeenshownthattheenzymenicotinamideadeninedinucleotidephosphateoxi-dase4(NOX4)playsakeyroleinosteoclastoge-nesis.
Micedeficientofthisenzyme,whichprodu-cesconstitutivelyROShaveahighbonemassandosteoclastmarkersdeficit;alsoinhumanbonesampleshighosteoclastactivityiscorrelatedwithincreasedactivityofNOX463.
Furthermore,itisnotedthatinsituationsofincreasedROSassocia-tedwithexperimentalDM,aremixedresults.
Whilesomeauthorshaveobservedanincreaseinosteoclastactivity64,ithasbeensuggestedthatcouldberelatedtothegreaterseverityofDM65,however,otherDMmodels,osteoclasticactivityisreduced66.
Infact,studiesusingmurineosteoclastspre-incubatedinthepresenceofhighglucoseappeartoconfirmtheirinhibitoryeffectonosteo-clasts67.
Thus,differencesinthedegreeofDM,strainandageoftheanimal,couldcontributetothevaryinglevelsofboneresorptionobservedindifferentmodels65,68.
PossibleoxidativestresstherapiesinsenileosteoporosisThedevelopmentofnewanabolictherapiesforoste-oporosisthatcombineincreasedbonemasswithitsabilitytoneutralizetheharmfuleffectsofoxidativestressisofgreatinterest.
Anintuitiveapproachtopreventbonelosswithagewouldbebasedontheantioxidantadministration.
However,itpointedoutthatclassicantioxidants,suchastheCATorN-acetylcysteine,exertundesirableeffectsonbonetis-sueasauthenticanti-osteoclastogenicactasagentsinterferingwithboneremodeling69.
Inaddition,suchagentsinhibitthecanonicalWnt/beta-cateninwhoseactivationisvitallyimportantformaintainingboneformation,partlybyinducingtheseizureofactiva-tingtheproteindisheveledbytheregulatoryproteinredoxbalance,nucleoredoxin70.
Recently,theboneanaboliceffecthasbeenasso-ciatedwithintermittentadministrationofparathyroidhormone(PTH)withitsstressoxidativeproperties,suchasthedecreaseintheamountofROS,inhibitionofphosphorylationofp66shcadaptorproteinandincreasingtheamountoftotalglutathione69.
TheadvantageofthistreatmentwithPTHversustheclas-Figure1.
GenerationandcelldamagecausedbyexcessROS.
ROSgenerationisaconsequenceofaerobicmeta-bolisminthemitochondrialrespiratorychain.
EnzymessuchasSOD,glutathionereductaseCATand/glutathioneperoxidasesystemareresponsibleformaintainingphysiologicallevelsofROS.
However,whenthisbalancedecompensatesexcessROSsynthesis,celldamagemayoccurleadingtoapoptosisofosteocytesandosteoblastsandincreasedosteoclastactivityIncreaseosteoclastactivityIncreaseboneresorptionApoptosisosteoblasts/osteocytesLipidperoxidationDamagetoDNA/proteinIncreaseofp-p66shcROSgenerationanddetoxificationMitochondriaDreeelectron+O2CatalaseGRaseGSHGSSGGPxSODH2O+O2H2O2H2OO2.
–OH.
–……REVIEW/RevOsteoporosMetabMiner.
2016;8(4):138-146142sicantioxidantsdeterminesitsstimulatoryactionofboneremodeling,withapredominanceofbonefor-mationinpartthroughitsinteractionwiththeWnt/beta-catenin(Figure2).
Inthiscontext,invitrotestinghasbeenshownthattheN-terminal(1-36)(homologouswithPTH)andC-terminal(107-109)ofthePTH-relatedprotein(PTHrP)fragmentsareabletocounteractoxidativestressinducedbyH2O2inosteoprogenitorcellsrelativetotheirosteogenicaction52,71.
Invitrostudiesandanimalmodelssuggestthatresveratrol,acompoundbifenilicgroupofpoly-phenolicantioxidantspresentintheskinofgrapesandotherfruits72,73,couldbeapotentialanti-osteo-poroticagent.
Thiscompoundincreasestheprolife-rationanddifferentiationofosteoblastinthepre-MC3T3-E1mouseinvitro73.
Furthermore,adminis-teringresveratroltomesenchymalcellsderivedfromhumanembryonicstemcellshasbeenshowntoinducetheexpressionofmatureRunx274diffe-rentiation75andosteoblasts.
ThismechanismofactionofresveratrolappearstobemediatedbySIRT1deacetylationactivationwhichincreasesFoxO3aexpressionandcomplexformationwithresveratrol,increasingRunx2expression(Figure3).
SIRT1couldalsoincreasetheactivityofRunx2directlybydeacetylationofthistranscriptionfactorinpre-osteoblastcells.
Inrecentresearchintoolderrats,administeringresveratrol(10mg/kgdailyfor10weeks)hasbeenshowntoimprovebonequa-lityandbonebiomechanicalpropertiesoftheoste-oporoticbone76.
Althoughthesepre-clinicalresultsarepromising,therearestillnoharddatatocon-firmtheefficacyofresveratrolinsenileosteoporo-sisinhumans.
However,ofnoteisarecentstudyconductedinobeseandosteopenicpatients,inwhichoraladministrationofresveratrol(1gdailyfor16weeks)significantlyincreasedbonemass,andtheamountofbonealkalinephosphatase,comparedtotheplacebogroup77.
Recentreportsindicatethatmicedefi-cientinSIRT6,anotherdeacetylaserelatedtotheresponsetooxidativestress,presentanosteoporoticphenotypeatanearlyage.
TheabsenceofSIRT6isassociatedwithoverex-pressionofRunx2,osterixandOPGaswellastheincreasedWntpathwayinhibitor,Dickkopf1,whichleadstoadeficitofosteoblastandosteoclastmaturation78.
ThesedatasuggestthatSIRT6couldbeatherapeutictargetininvolu-tionalosteoporosis.
Furthermore,glucocorticoidexcessalsoinducesoxidativestress.
Inthissituation,theoxidativestressobservedinplasmareticulumcanbereversedbytranslationinitiationfactor2αphosphorylation,whichdisruptsproteintranslation.
Adephosphorylationinhibitorcom-pound,salubrinal,hasrecentlybeenshowntopreventdeficitmineralizationofosteoblaststre-atedwithglucocorticoidsinvitroaswellasosteoblastandosteocyteapoptosisinanosteo-poroticmousemodelbyprednisoloneadminis-tration79.
ConclusionsTheprogressiveagingofthepopulationinthedeve-lopedworldleadstoincreasedmusculoskeletaldisor-ders,includingosteoporosis.
Osteoporosisandincre-asedfragilityoftheelderlypopulationareasocio-economicchallengeofthefirstmagnitude.
Differentfactorscontributetobonelossintheelderly,amongwhichstandsoutasacommonelementincreasedoxidativestress(Figure4).
Thus,reducingoxidativestresscouldbeausefultooltocombatinvolutionalosteoporosis.
However,thefactthatoxidativestresscompoundscouldinterferewiththeboneremode-lingorkeyanabolicpathwaysforboneformation,suchastheWntsignalingpathway,requirescertainconsiderationspriortotherapeuticuse.
WemustalsotakeintoaccountthephysiologicalroleofROS,whichactassecondarymessengersofmanymetabo-licpathways;thereforeitsuncontrolledinhibitioncouldleadtounwantedsideeffectsinbonecells.
Furtherresearchisneededtodeterminethetrueeffectofantioxidanttherapiesandappropriatedosingschedulestoavoiddeleteriousactiononboneremo-deling.
Takingintoaccounttheseconsiderations,the-rapiesaimedatneutralizingoxidativestresstopre-ventoralterthecourseofinvolutionalosteoporosiswouldrepresentanobviousmedicalbreakthrough.
Competinginterests:Theauthorsdeclarenoconflictsofinterest.
Financing:ThisworkhasbeenfundedbyaidfromtheSpanishFoundationforBoneResearchandMineralMetabolism(GrantFEIOMMTranslationalResearch2015),theInstituteCarlosIII(RD12/0043/0022,PI11/00449,RD06/0013/1002Figure2.
OsteogenicactionsofPTHthroughtheWnt/beta-cateninpathway.
PTHisabletodirectlyactivatetheWntpathwaybybindingofthetype1receptor(PTH1R)withcoreceptorWntproteins,therelatedreceptorlow-densitylipoprotein6(LRP6).
Furthermore,phosphorylationofAktactivationproducedbyPTH1RresultsinFoxOdegradation,whichfavorsbeta-cateninstabilizationPTHActiveWntIncreaseavailabilityβ-cateninOsteogenicactivationprogramFoxOdestructionAktβ-cateninPTH1RPTH1RLRP6REVIEW/RevOsteoporosMetabMiner.
2016;8(4):138-146143Health,RD12/0043/0018andRD12/0043/0008).
SP-NenjoyedbyaRETICEFcontract(RD06/0013/1002andRD12/0043/0008).
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