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PECAM-1 gene polymorphism (rs668) and subclinical markers of carotid atherosclerosis in patients with type 2 diabetes mellitus

PECAM-1 gene polymorphism (rs668) and subclinical markers of carotid atherosclerosis in patients... Theplateletendothelialcelladhesionmolecule1 (PECAM-1)playsanimportantroleinmanyinflammatoryprocesses,includingthedevelopmentofatherosclerosis.Polymorphismrs668ofthePECAM-1 gene(373C/G)isfunctional,anditwasreportedtobe associatedwithincreasedserumlevelsofPECAM-1. Weinvestigatedtheassociationbetweenthers668 polymorphismofPECAM-1andsubclinicalmarkersofcarotidatherosclerosisinsubjectswithtype2 diabetesmellitus(T2DM).FivehundredandninetyfiveT2DMsubjectsand200controlsubjectswere enrolled.Thecarotidintima-mediathickness(CIMT) andplaquecharacteristics(presenceandstructure) were assessed ultrasonographically. Biochemical analyseswereperformedusingstandardbiochemical methods.Geno-typingofthePECAM-1genepolymorphism(rs668)wasperformedusingKASParassays.Thecontrolexaminationswereperformed3.8± 0.5yearsaftertheinitialexamination.HigherCIMT wasfoundinpatientswithT2DMincomparisonwith subjectswithoutT2DM.Statisticallysig-nificantly fasterprogressionoftheatheroscleroticmarkerswas GeneralHospitalRakican,MurskaSobota,Slovenia InstituteofHistologyandEmbryology,FacultyofMedicine, UniversityinLjubljana,Ljubljana,Slovenia 3 GeneralHospitalSlovenjGradec,SlovenjGradec,Slovenia 4 ZdravstveniZavodReschner,Ljubljana,Slovenia 5 CardiologyOutpatientDepartmentMedicalCenterMedicor, Ljubljana,Slovenia showninsubjectswithT2DMincomparisonwith thecontrolgroup.Whenadjustedtootherriskfactors,thers668GGgenotypewasassociatedwithan increasedriskofcarotidplaquesinsubjectswith T2DM.Weconcludedthatourstudydemonstrated aminoreffectofthers668PECAM-1onmarkersof carotidatherosclerosisinsubjectswithT2DM. Keywords:Associationstudy;carotidatherosclerosis;Plateletendothelialcelladhesionmolecule 1(PECAM-1);rs668polymorphism;type2diabetes mellitus(T2DM). INTRODUCTION Theleukocyteadhesionandtheirtransendothelialmigrationplayanimportantroleintheinitial phaseofatherogenesis[1].Processesareregulatedby varioustypesofadhesionmolecules,suchasplatelet endothelialcelladhesionmolecule1(PECAM-1), intercellular adhesion molecule 1 (ICAM-1) and vascularcelladhesionmolecule1(VCAM-1).The plasmalevelofadhesionmoleculesiselevatedin individualswithatherosclerosis[2-4]. Plateletendothelialcelladhesionmolecule1, alsocalledCD31,isa130kDmemberoftheimmunoglobulinsuperfamily,consistingofsixextracellularimmunoglo-bulin-likedomains,onetransmembranedomain,andonecytoplasmicdomain.Their expressiontakesplaceonthesurfaceofcirculating platelets,monocytes,neutrophilsandselectedTcells [5,6].ThePECAM-1isasignalingmoleculethat playsdiverserolesinvascularbiology,including modulationofplateletfunction[7,8],angiogenesis [9],vasculogenesis[10],integrinregulation[11], T-cellandB-cellactivation[12]andmediationof leukocytemigrationacrosstheendothelium[13]. ThePECAM-1geneislocatedattheendofthe longarmofthechromosome17(17q23).Previous studieshavereportedtheexistenceof11differentsingle nucleotidepolymorphisms(SNPs)ofthePECAM-1 gene.Threeofthemhavebeendescribedthatencode aminoacidsubstitutionsinthePECAM-1molecule.A mutationinthePECAM-1geneinexon3atposition +373involvesaC>Gsubstitution,causingaleucineto valinesubstitutionatposition125(rs668)[14]. TheinteractionoractivationofthePECAM-1take placeviahomophilicbindingwithitsfirstextracellular Ig-likedomains[15,16].Thispolymorphismmightaffectthehomophilicbindingcapabilityandinfluence individualsusceptibilitytothedevelopmentofatherosclerosis.Theassociationbetweenthers668PECAM-1 polymorphismandcardiovasculardiseasewasstudied inCaucasians[17-19],Japanese[20]andChinese[21], butnoclearanswerontheassociationbetweenthers668 polymorphismofPECAM-1andthedevelopmentof cardiovasculardiseasescouldbeprovided. Plateletendothelialcelladhesionmolecule1 isimportantinthedetectionofmechanoreception (mechanicalshearforce)andmechanotransduction (conversionintochemicalsignals)bytheendothelium [22,23].Atheroscleroticlesion development occursatsitesofthevesselwhereflowandshear stress conditions are disturbed [24]. Pulsatile or oscillatoryshearstressesinduceproinflammatory geneexpression[25].Usingthemousemodel,the effectofPECAM-1deficiency(doubleknock-out micemodelwithoutthepresenceofthePECAM-1 gene)onthedevelopmentofatherosclerosis.They reportedreducedatheroscleroticlesionsindouble knock-outmicemodels[21,25].Thepurposeofthis studywastoinvestigateanassociationbetweenthe rs668(+373C/G)polymorphismofthePECAM-1 geneandsubclinicalmarkersofcarotidatherosclerosisinpatientswithtype2diabetesmellitus(T2DM). PATIENTS AND METHODS Thisstudyincluded595consecutivesubjects withT2DM,admittedtothediabetesoutpatientclinicsofthegeneralhospitalsatMurskaSobotaand SlovenjGradec,Slovenia,andfromtheoutpatient departmentattheMedicalCenterMedicor,Ljubljana, Slovenia.Theinclusioncriteriaforthecontrolgroup wastheabsenceofT2DM,andconsistedofemployeesoftheGeneralHospitalMurskaSobota,Slovenia. AnotherinclusioncriteriaforthesubjectswithT2DM andforthesubjectsinthecontrolgroupwastheage from40to70.Theexclusioncriteriaforsubjects withT2DMandforthesubjectsinthecontrolgroup wasahistoryofeithermyocardialinfarction(MI)or ischemicstroke.Thestudyprotocolwasapprovedby theSloveneMedicalEthicsCommittee(98/08/10). Thepatientsandcontrolsubjectswereenrolledand followedintheperiodfrom2008to2014. PatientswereclassifiedashavingT2DMaccordingtothecurrentreportoftheAmericanDiabetesAssociation[26].Afterinformedconsentwasobtained fromthepatients,adetailedinterviewwasconducted concerningsmokinghabits,thedurationandtreatmentofdiabetes,arterialhypertension,andhyperlipidemia.Patientswereaskedwhethertheywere smokersatthetimeofrecruitment(currentsmoker). SubjectswithT2DMwithsystolicbloodpressure 140.0mmHgordiastolicbloodpressure85.0mm Hgand/orsubjectswhoweretakingantihypertensive drugswereconsideredtobehypertensive. Allultrasoundexaminationswereperformedby twoexperienceddoctorsblindedtotheparticipants' diabetesstatus.Thecarotidintima-mediathickness (CIMT),definedasthedistancefromtheleading edgeofthelumen-intimainterfacetotheleadingedge ofthemedia-adventitiainterface,wasmeasuredas previouslydescribed[27].Plaquesweredefinedasa focalintima-mediathickening,anddividedintofive typesaccordingtotheirechogenic/echolucentcharacteristics,aspreviouslydescribed[27].Theinterobserverreliabilityforcarotidplaquecharacterizationwasfoundtobesubstantial(=0.64,p<0.001). AfterthepatientswithT2DMandsubjectswithoutT2DM(controlgroup)wereenrolled,theywere prospectivelyfollowed-upforafewyears.Fromthe groupwithT2DM,426respondedandparticipatedin thecontrolultrasoundexaminationoftheneckartery, whereas,132re-spondedfromthegroupofsubjects withoutT2DM;3.8±0.5yearspassedbetweenthe firstandthecontrolultrasoundexamination. Biochemical Analyses.Fastingbloodsamples forbiochemicalanalysiswerecollectedtwicepatients:uponenrollmentanduponfollow-upaftera fewyears.Analysesweremadeatthehospitalac- creditedlaboratory.Thefollowingparameterswere determined: total cholesterol, triglycerides, highdensitylipoprotein(HDL),low-densitylipoprotein (LDL),bloodsugarandhigh-sensitiveC-reactive protein(hsCRP). Genotyping.GenomicDNAwasextractedfrom 100µLofwholebloodusingaFlexiGeneDNAisolationkit(QiagenGmbH,Hilden,Germany),inaccordancewiththerecommendedprotocol.Thers668 polymorphismofthePECAM-1genewasdetermined withtheKASParassay(LGCGenomicsLtd.,Hoddesdon,Hertfordshire,UK)system. Statistical Analyses.Continuousvariablesthat werenormallydistributed,werereportedintheform ofmean±standarddeviation(SD).Variablesthat werenotnormallydistributed,werepresentedinthe formofmedian(inter-quartilerange).Thenormality ofdistributionofcontinuousvariableswasexamined usingtheKolmogorov-Smirnov test.Weusedthe Student'sttestortheanalysisofvariance(ANOVA) tocomparethenumericalvaluesofthecontinuous variables,toidentifythedistributionofvariables.If thevariableswereasymmetricallydistributed,we usedtheMann-WhitneyUtestortheKruskal-Wallis Htest.The2testwasusedtocomparethefrequency'scategoricalvariables,statisticalevaluationof differencesinthefrequenciesofdifferentallelesand genotypesbetweenthetwogroups,aswellasinthe caseofdeterminingtheHardy-Weinbergequilibrium. ThePearsonanalyseswasperformedtoexamine thecorrelationbetweentheindependentvariables. TheresultsshowedahighdegreeofcorrelationbetweentheserumlevelsoftotalandLDLcholesterol (r=0.86;p<0.001),aswellassystolicanddiastolic bloodpressure(r=0.65;p<0.001).Inthecaseofa highdegreecorrelationbetweentwovariables,only onevariablefromeachpairwasincludedinthemultivariatestatisticalmodels. Thechangeinthevalueofultrasoundmarkers ofcarotidarteryatherosclerosiswascalculatedby deductingthevaluesmeasuredattwoultrasoundexaminations.Thecriteriaforastatisticallysignificant differencewasapvalueoflessthan0.05.Toreduce thepossibilityoferrorduetothesmallnumberofsubjects,weusedtheBonferronicorrection.Allstatistical analyseswereperformedusingtheStatisticalPackage fortheSocialSciences(SPSS)computerprogramfor Windows,version20(SPSSInc.,Chicago,IL,USA). RESULTS TheclinicalcharacteristicsofsubjectswithT2DM andcontrolsubjectsareshowninTable1.Patientswith T2DMhadagreaterwaistcircumferenceandthere Table 1.Initialclinicalandbiochemicalcharacteristicsofpatientswithtype2diabetesmellitusandthecontrolgroup. Parameters Age(mean±SD) Sex:M(%);F(%) DurationofT2DM Smokers:M(%);F(%) Waistcircumference(cm) BMI(kg/m2) Systolicpressure(mmHg) Diastolicpressure(mmHg) Fastingglucose(mmol/L) HbA1c(%) Totalcholesterol(nmol/L) HDLcholesterol(mmol/L) LDLcholesterol(mmol/L) Triglycerides(mmol/L) HighsensitivityCRP(mg/L) CIMT(µm) Patients with T2DM (n = 529) 61.38±9.65 M:338(56.8);F:191(43.2) 11.5±7.88 M:30(8.9);F:23(12.0) 108.65±12.88 30.96±4.74 146.98±19.98 85.75±11.62 8.04±2.57 7.89±3.56 4.70±1.19 1.19±0.35 2.63±0.94 1.90(1.20-2.70) 2.20(1.00-4.30) 1013.00±208.00 Control Group (n = 200) 60.07±9.18 M:92(46.0);F:108(54.0) ­ M:19(20.6);F:15(13.9) 93.31±13.18 27.90±4.42 143.30±16.60 84.70±11.60 5.27±0.87 4.79±0.29 5.36±1.08 1.43±0.37 3.24±0.98 1.30(0.90-1.90) 1.30(0.80-2.70) 979.00±141.00 p Value 0.07 0.008 ­ 0.002 <0.001 0.16 0.86 0.19 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.030 T2DM:type2diabetesmellitus;BMI:bodymassindex;HDL:high-densitylipoprotein;LDL:low-densitylipoprotein;CRP:C-reactiveprotein; CIMT:carotidintima-mediathickness. Table 2.Changesinultrasoundmarkersofatherosclerosisatcarotidarteryinpatientswithtype2diabetesmellitusandthe controlgroupbetweentheinitialandcontrolultrasoundexaminations. Parameters AnnualCIMTincrement(µm/year) numberofplaquesegments sumoftheplaquethickness(mm) Patients with T2DM (n = 426) 20.33(11.74-29.86) 2.00(1.00-3.00) 5.40(2.40-7.05) Control Group (n = 137) 12.83(8.82-20.66) 1.50(0.70-2.20) 3.64(2.88-5.48) 0.50 0.00 0.00 0.02 CIMT:carotidintima-mediathickness;:variablevaluechangesduringtheobservationperiod,expressedasapercentageofbaselinevalues. Table 3.Comparisonofultrasoundmarkersofthecarotidarteryatherosclerosisinpatientswithtype2diabetesmellitus accordingtothePECAMgenepolymorphism373C/G(rs668)genotypesatthebeginningofthestudy. Parameters CIMT(µm) Numberofsegmentswithplaques Sumoftheplaquethickness(mm) Presenceofplaques:[+] [­] Presenceofunstableplaques:[+] [­] CIMT:carotidintima-mediathickness. CC Genotype 1041.00±198.00 2.56±1.65 8.14±4.77 149.00(85.60) 25.00(14.40) 86.00(57.70) 63.00(42.30) CG Genotype 990.00±187.00 2.39±1.72 7.99±5.23 256.00(84.50) 47.00(15.50) 144.00(56.30) 112.00(43.70) GG Genotype 988.00±211.00 2.51±1.54 7.66±4.02 94.00(79.70) 24.00(20.30) 58.00(61.70) 36.00(38.30) p value 0.19 0.36 0.16 0.36 0.66 Table 4.Changesinultrasoundmarkersofcarotidarteryatherosclerosisinpatientswithtype2diabetesmellitusbetween thefirstandthesecondultrasoundexaminationofthecarotidarteriesaccordingtothePECAMgenepolymorphism 373C/G(rs668)genotypes. Parameters AnnualCIMTincrement(µm/year) numberofplaquesegments sumoftheplaquethickness(mm) CC Genotype 20.34(11.64-28.04) 2.00(0.50-2.50) 7.30(3.85-8.70) CG Genotype 20.69(7.14-32.28) 2.00(1.00-3.00) 5.45(2.30-9.20) GG Genotype 14.28(10.71-35.97) 2.00(2.00-3.00) 4.40(1.40-8.42) p Value 0.71 0.74 0.38 CIMT:carotidintima-mediathickness;:variablevaluechangesduringtheobservationperiod,expressedasapercentageofbaselinevalues. weremoresmokerscomparedtothecontrolgroup. There were no statistically significant differences betweenpatientswithT2DMandcontrolsinother clinicalcharacteristics[age,bodymassindex(BMI), systolicanddiastolicpressure].AbiochemicalexaminationofpatientswithT2DMshowedstatistically significanthigherlevelsoffastingglucose,HbA1c, totalcholesterol,HDL,LDL,triglycerideandhsCRP comparedwiththecontrolgroup(Table1).Moreover, higherCIMTwasfoundinpatientswithT2DMin comparisonwithsubjectswithoutT2DM(Table1). Theultrasoundexaminationofthecarotidarterywasperformedatthetimeofenrollmentinthe study,and3.8±0.5yearsaftertheinitialexamination. Changesintheprogressionofatheroscleroticmarkers (changeinannualCIMTincrease,changeinthenumberofplaquesegmentsandchangeinthesumofthe plaquethickness)betweensubjectswithT2DMand thecontrolgroupareshowninTable2.Statistically significantlyfasterprogressionoftheatherosclerotic markerswasshowninsubjectswithT2DMincomparisonwiththecontrolgroup(Table2). Thedistributionofgenotypesinthepopulation ofpatientswithT2DMwasinHardy-Weinbergequilibrium(T2DM:2=0.45;p=0.50;controlgroup: 2=1.46;p=0.23).Table3outlinesdifferencesin theultrasoundmarkersofcarotidarteryatherosclerosis(initialultrasoundexamination)inpatientswith T2DMwithregardtothers668genotypes.Thedifferenceswerenotstatisticallysignificantwithregard tors668genotypes(Table3).Wedidnotdemonstrate statisticallysignificantdifferencesinthemarkersof subclinicalcarotidatherosclerosisbetweendifferent genotypesinsubjectswithT2DM(Table4). Table5showstherelationbetweenthers668and theincidenceofeitherplaquesorunstableplaques Table 5.RelationshipofthePECAMgenepolymorphism373C/G(rs668)genotypestothepresenceofplaques/unstable plaquesofthecarotidarteriesinpatientswithtype2diabetesmellitusatthebeginningofthestudy. Presence of Plaques Parameters Hypertension(0=no;1=yes) Systolicpressure(mmHg) SerumLDL(mmol/L) SerumHDL(mmol/L) HbA1c(%) CG allele GG allele OR (95% CI) 1.88 0.24 1.41 0.11 0.89 1.03 1.18 p Value 0.35 0.68 0.41 0.02 0.03 0.49 0.03 Presence of Unstable Plaques OR (95% CI) 1.38 0.26 1.34 0.31 1.23 0.72 0.68 p Value 0.52 0.32 0.25 0.34 0.43 0.17 0.51 OR(95%CI):oddsratio(95%confidenceinterval);LDL:low-densitylipoprotein;HDL:high-densitylipoprotien;allmodelswereadjustedbyage, sex,smokinghabitsandtreatmentwithstatins.ThereferencegrouparehomozygotesfortheCallele(theCCgenotype). insubjectswithT2DM.Whenadjustedtootherrisk factors,thers668GGgenotypewasassociatedwith anincreasedriskofcarotidplaquesinsubjectswith T2DM(Table5). DISCUSSION Inthepresentstudy,wedemonstratedanassociationbetweenthers668GGgenotypeandcarotid arteryplaqueincidenceinpatientswithT2DMwhen adjustedtootherriskfactors.Ontheotherhand,we didnotfindanyeffectofthers688genotypeonthe progressionofatherosclerosisinpatientswithT2DM. Ourstudyisthefirstdemonstratinganassociationbetweenthers668PECAM-1andthepresenceof carotidplaquesinpatientswithT2DM.Inourstudy, weobservedagreaternumberofplaquesinsubjects withtheGGrs668genotype.Theimportanceofthe PECAM-1geneinthepathogenesisofatherosclerosis wasdemonstrated[21,25].Thedecreaseinareaswith atheroscleroticlesioninPECAM-1doubleknock-out micewasreported[21,25]. Anassociationbetweenthers668PECAM-1and car-diovasculardisorderswasreportedseveraltimes, butnotinallstudies[29-33].Similarly,anassociationbetweenthers668PECAM-1geneandeitherthe increasedlevelsofsolublePECAM-1orischemic strokewasfoundintheChinesepopulation[21].An associationbetweenthers668PECAM-1geneand increasedlevelsofsolublePECAM-1wasconfirmed inthesettingofpatientswithacuteMI[28]. Similarly,Fanget al.[29]demonstratedanassociationbetweenthers668PECAM-1geneandeither increasedlevelsofsolublePECAM-1ortheseverity ofcoronaryarterydiseaseintheAsianIndianpopulationinSingapore.Reschneret al.[17]reportedan associationbetweenthePECAM-1rs668(theCC genotype)andMIinCaucasianswithT2DM.Finally, theeffectofseveralpolymorphismsofthePECAM-1 geneoncardiovascular diseasewasconfirmedby Listi et al.[30]intheNorthernItalianpopulation. Thesefindingswerenotconfirmedinthegeneral populationfromGermany,asrs688wasnotfound tobeanindependentriskfactorforcoronaryartery disease(CAD)[31]. Inarecentlypublishedmeta-analysisof15studies,including7636subjects,noassociationbetween thers668PECAM-1andcardiovasculardiseaseswas demonstrated[35].Moreover,theprogressionofsubclinicalmarkersofcarotidatherosclerosiswasstatisticallysignificantlyfasterinsubjectswithT2DM incomparisonwithsubjectswithoutT2DM.This findingisinaccordancewithexpectationsofother researchersaswell[32,33]. Ourstudyhassomelimitationsduetoitsactual design(cross-sectionaldesignattheenrollmentof subjectswithT2DMandcontrolsubjects)andrelativelysmallsamplesize(i.e.,lessthan1000subjects withT2DM),howeverthestudywasappropriately poweredtodetectthedifferencesinsubclinicalmarkersofcarotidatherosclerosisuponenrollmentaswell asduringfollow-up.Thelackoftheeffectofrs688 ontheprogressionofatherosclerosismightbedueto arathershortintervalbetweenthefirstandcontrol examinations(3.8±0.5years).Moreover,itisimpossibletoexcludetheimpactofinteractionswithother potentiallyrelevantvariablesonthedevelopmentof atherosclerosis.Thesecondlimitationofthestudy isthelackofanintermediatephenotype,i.e.,serum PECAMlevels.Wepresumethattheeffectofrs668 is viaincreasedserumPECAMlevels. Inconclusion,ourstudydemonstratedaminor effectofthers668PECAM-1onthemarkersofcarotidatherosclerosisinsubjectswithT2DM,asthe GGgenotypeofthers668PECAM-1wasassociated withahigherincidenceofcarotidplaquesinsubjects withT2DM.Withthatkindofassociationsestablishedingeneticstudies,wepresumedthatwemight predictthegeneticriskofcarotidatherosclerosisin subjectswithT2DM. ACKNOWLEDGMENTS TheauthorsthankMrs.BrinaBeskonik,B.A., JezicekBrinaBeskovniks.p.,Portoroz-Portorose, Slovenia,forrevisingtheEnglish. Declaration of Interest.Theauthorsreportno conflictsofinterest.Theauthorsaloneareresponsible forthecontentandwritingofthisarticle. 8. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Balkan Journal of Medical Genetics de Gruyter

PECAM-1 gene polymorphism (rs668) and subclinical markers of carotid atherosclerosis in patients with type 2 diabetes mellitus

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de Gruyter
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1311-0160
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10.1515/bjmg-2016-0008
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Abstract

Theplateletendothelialcelladhesionmolecule1 (PECAM-1)playsanimportantroleinmanyinflammatoryprocesses,includingthedevelopmentofatherosclerosis.Polymorphismrs668ofthePECAM-1 gene(373C/G)isfunctional,anditwasreportedtobe associatedwithincreasedserumlevelsofPECAM-1. Weinvestigatedtheassociationbetweenthers668 polymorphismofPECAM-1andsubclinicalmarkersofcarotidatherosclerosisinsubjectswithtype2 diabetesmellitus(T2DM).FivehundredandninetyfiveT2DMsubjectsand200controlsubjectswere enrolled.Thecarotidintima-mediathickness(CIMT) andplaquecharacteristics(presenceandstructure) were assessed ultrasonographically. Biochemical analyseswereperformedusingstandardbiochemical methods.Geno-typingofthePECAM-1genepolymorphism(rs668)wasperformedusingKASParassays.Thecontrolexaminationswereperformed3.8± 0.5yearsaftertheinitialexamination.HigherCIMT wasfoundinpatientswithT2DMincomparisonwith subjectswithoutT2DM.Statisticallysig-nificantly fasterprogressionoftheatheroscleroticmarkerswas GeneralHospitalRakican,MurskaSobota,Slovenia InstituteofHistologyandEmbryology,FacultyofMedicine, UniversityinLjubljana,Ljubljana,Slovenia 3 GeneralHospitalSlovenjGradec,SlovenjGradec,Slovenia 4 ZdravstveniZavodReschner,Ljubljana,Slovenia 5 CardiologyOutpatientDepartmentMedicalCenterMedicor, Ljubljana,Slovenia showninsubjectswithT2DMincomparisonwith thecontrolgroup.Whenadjustedtootherriskfactors,thers668GGgenotypewasassociatedwithan increasedriskofcarotidplaquesinsubjectswith T2DM.Weconcludedthatourstudydemonstrated aminoreffectofthers668PECAM-1onmarkersof carotidatherosclerosisinsubjectswithT2DM. Keywords:Associationstudy;carotidatherosclerosis;Plateletendothelialcelladhesionmolecule 1(PECAM-1);rs668polymorphism;type2diabetes mellitus(T2DM). INTRODUCTION Theleukocyteadhesionandtheirtransendothelialmigrationplayanimportantroleintheinitial phaseofatherogenesis[1].Processesareregulatedby varioustypesofadhesionmolecules,suchasplatelet endothelialcelladhesionmolecule1(PECAM-1), intercellular adhesion molecule 1 (ICAM-1) and vascularcelladhesionmolecule1(VCAM-1).The plasmalevelofadhesionmoleculesiselevatedin individualswithatherosclerosis[2-4]. Plateletendothelialcelladhesionmolecule1, alsocalledCD31,isa130kDmemberoftheimmunoglobulinsuperfamily,consistingofsixextracellularimmunoglo-bulin-likedomains,onetransmembranedomain,andonecytoplasmicdomain.Their expressiontakesplaceonthesurfaceofcirculating platelets,monocytes,neutrophilsandselectedTcells [5,6].ThePECAM-1isasignalingmoleculethat playsdiverserolesinvascularbiology,including modulationofplateletfunction[7,8],angiogenesis [9],vasculogenesis[10],integrinregulation[11], T-cellandB-cellactivation[12]andmediationof leukocytemigrationacrosstheendothelium[13]. ThePECAM-1geneislocatedattheendofthe longarmofthechromosome17(17q23).Previous studieshavereportedtheexistenceof11differentsingle nucleotidepolymorphisms(SNPs)ofthePECAM-1 gene.Threeofthemhavebeendescribedthatencode aminoacidsubstitutionsinthePECAM-1molecule.A mutationinthePECAM-1geneinexon3atposition +373involvesaC>Gsubstitution,causingaleucineto valinesubstitutionatposition125(rs668)[14]. TheinteractionoractivationofthePECAM-1take placeviahomophilicbindingwithitsfirstextracellular Ig-likedomains[15,16].Thispolymorphismmightaffectthehomophilicbindingcapabilityandinfluence individualsusceptibilitytothedevelopmentofatherosclerosis.Theassociationbetweenthers668PECAM-1 polymorphismandcardiovasculardiseasewasstudied inCaucasians[17-19],Japanese[20]andChinese[21], butnoclearanswerontheassociationbetweenthers668 polymorphismofPECAM-1andthedevelopmentof cardiovasculardiseasescouldbeprovided. Plateletendothelialcelladhesionmolecule1 isimportantinthedetectionofmechanoreception (mechanicalshearforce)andmechanotransduction (conversionintochemicalsignals)bytheendothelium [22,23].Atheroscleroticlesion development occursatsitesofthevesselwhereflowandshear stress conditions are disturbed [24]. Pulsatile or oscillatoryshearstressesinduceproinflammatory geneexpression[25].Usingthemousemodel,the effectofPECAM-1deficiency(doubleknock-out micemodelwithoutthepresenceofthePECAM-1 gene)onthedevelopmentofatherosclerosis.They reportedreducedatheroscleroticlesionsindouble knock-outmicemodels[21,25].Thepurposeofthis studywastoinvestigateanassociationbetweenthe rs668(+373C/G)polymorphismofthePECAM-1 geneandsubclinicalmarkersofcarotidatherosclerosisinpatientswithtype2diabetesmellitus(T2DM). PATIENTS AND METHODS Thisstudyincluded595consecutivesubjects withT2DM,admittedtothediabetesoutpatientclinicsofthegeneralhospitalsatMurskaSobotaand SlovenjGradec,Slovenia,andfromtheoutpatient departmentattheMedicalCenterMedicor,Ljubljana, Slovenia.Theinclusioncriteriaforthecontrolgroup wastheabsenceofT2DM,andconsistedofemployeesoftheGeneralHospitalMurskaSobota,Slovenia. AnotherinclusioncriteriaforthesubjectswithT2DM andforthesubjectsinthecontrolgroupwastheage from40to70.Theexclusioncriteriaforsubjects withT2DMandforthesubjectsinthecontrolgroup wasahistoryofeithermyocardialinfarction(MI)or ischemicstroke.Thestudyprotocolwasapprovedby theSloveneMedicalEthicsCommittee(98/08/10). Thepatientsandcontrolsubjectswereenrolledand followedintheperiodfrom2008to2014. PatientswereclassifiedashavingT2DMaccordingtothecurrentreportoftheAmericanDiabetesAssociation[26].Afterinformedconsentwasobtained fromthepatients,adetailedinterviewwasconducted concerningsmokinghabits,thedurationandtreatmentofdiabetes,arterialhypertension,andhyperlipidemia.Patientswereaskedwhethertheywere smokersatthetimeofrecruitment(currentsmoker). SubjectswithT2DMwithsystolicbloodpressure 140.0mmHgordiastolicbloodpressure85.0mm Hgand/orsubjectswhoweretakingantihypertensive drugswereconsideredtobehypertensive. Allultrasoundexaminationswereperformedby twoexperienceddoctorsblindedtotheparticipants' diabetesstatus.Thecarotidintima-mediathickness (CIMT),definedasthedistancefromtheleading edgeofthelumen-intimainterfacetotheleadingedge ofthemedia-adventitiainterface,wasmeasuredas previouslydescribed[27].Plaquesweredefinedasa focalintima-mediathickening,anddividedintofive typesaccordingtotheirechogenic/echolucentcharacteristics,aspreviouslydescribed[27].Theinterobserverreliabilityforcarotidplaquecharacterizationwasfoundtobesubstantial(=0.64,p<0.001). AfterthepatientswithT2DMandsubjectswithoutT2DM(controlgroup)wereenrolled,theywere prospectivelyfollowed-upforafewyears.Fromthe groupwithT2DM,426respondedandparticipatedin thecontrolultrasoundexaminationoftheneckartery, whereas,132re-spondedfromthegroupofsubjects withoutT2DM;3.8±0.5yearspassedbetweenthe firstandthecontrolultrasoundexamination. Biochemical Analyses.Fastingbloodsamples forbiochemicalanalysiswerecollectedtwicepatients:uponenrollmentanduponfollow-upaftera fewyears.Analysesweremadeatthehospitalac- creditedlaboratory.Thefollowingparameterswere determined: total cholesterol, triglycerides, highdensitylipoprotein(HDL),low-densitylipoprotein (LDL),bloodsugarandhigh-sensitiveC-reactive protein(hsCRP). Genotyping.GenomicDNAwasextractedfrom 100µLofwholebloodusingaFlexiGeneDNAisolationkit(QiagenGmbH,Hilden,Germany),inaccordancewiththerecommendedprotocol.Thers668 polymorphismofthePECAM-1genewasdetermined withtheKASParassay(LGCGenomicsLtd.,Hoddesdon,Hertfordshire,UK)system. Statistical Analyses.Continuousvariablesthat werenormallydistributed,werereportedintheform ofmean±standarddeviation(SD).Variablesthat werenotnormallydistributed,werepresentedinthe formofmedian(inter-quartilerange).Thenormality ofdistributionofcontinuousvariableswasexamined usingtheKolmogorov-Smirnov test.Weusedthe Student'sttestortheanalysisofvariance(ANOVA) tocomparethenumericalvaluesofthecontinuous variables,toidentifythedistributionofvariables.If thevariableswereasymmetricallydistributed,we usedtheMann-WhitneyUtestortheKruskal-Wallis Htest.The2testwasusedtocomparethefrequency'scategoricalvariables,statisticalevaluationof differencesinthefrequenciesofdifferentallelesand genotypesbetweenthetwogroups,aswellasinthe caseofdeterminingtheHardy-Weinbergequilibrium. ThePearsonanalyseswasperformedtoexamine thecorrelationbetweentheindependentvariables. TheresultsshowedahighdegreeofcorrelationbetweentheserumlevelsoftotalandLDLcholesterol (r=0.86;p<0.001),aswellassystolicanddiastolic bloodpressure(r=0.65;p<0.001).Inthecaseofa highdegreecorrelationbetweentwovariables,only onevariablefromeachpairwasincludedinthemultivariatestatisticalmodels. Thechangeinthevalueofultrasoundmarkers ofcarotidarteryatherosclerosiswascalculatedby deductingthevaluesmeasuredattwoultrasoundexaminations.Thecriteriaforastatisticallysignificant differencewasapvalueoflessthan0.05.Toreduce thepossibilityoferrorduetothesmallnumberofsubjects,weusedtheBonferronicorrection.Allstatistical analyseswereperformedusingtheStatisticalPackage fortheSocialSciences(SPSS)computerprogramfor Windows,version20(SPSSInc.,Chicago,IL,USA). RESULTS TheclinicalcharacteristicsofsubjectswithT2DM andcontrolsubjectsareshowninTable1.Patientswith T2DMhadagreaterwaistcircumferenceandthere Table 1.Initialclinicalandbiochemicalcharacteristicsofpatientswithtype2diabetesmellitusandthecontrolgroup. Parameters Age(mean±SD) Sex:M(%);F(%) DurationofT2DM Smokers:M(%);F(%) Waistcircumference(cm) BMI(kg/m2) Systolicpressure(mmHg) Diastolicpressure(mmHg) Fastingglucose(mmol/L) HbA1c(%) Totalcholesterol(nmol/L) HDLcholesterol(mmol/L) LDLcholesterol(mmol/L) Triglycerides(mmol/L) HighsensitivityCRP(mg/L) CIMT(µm) Patients with T2DM (n = 529) 61.38±9.65 M:338(56.8);F:191(43.2) 11.5±7.88 M:30(8.9);F:23(12.0) 108.65±12.88 30.96±4.74 146.98±19.98 85.75±11.62 8.04±2.57 7.89±3.56 4.70±1.19 1.19±0.35 2.63±0.94 1.90(1.20-2.70) 2.20(1.00-4.30) 1013.00±208.00 Control Group (n = 200) 60.07±9.18 M:92(46.0);F:108(54.0) ­ M:19(20.6);F:15(13.9) 93.31±13.18 27.90±4.42 143.30±16.60 84.70±11.60 5.27±0.87 4.79±0.29 5.36±1.08 1.43±0.37 3.24±0.98 1.30(0.90-1.90) 1.30(0.80-2.70) 979.00±141.00 p Value 0.07 0.008 ­ 0.002 <0.001 0.16 0.86 0.19 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.030 T2DM:type2diabetesmellitus;BMI:bodymassindex;HDL:high-densitylipoprotein;LDL:low-densitylipoprotein;CRP:C-reactiveprotein; CIMT:carotidintima-mediathickness. Table 2.Changesinultrasoundmarkersofatherosclerosisatcarotidarteryinpatientswithtype2diabetesmellitusandthe controlgroupbetweentheinitialandcontrolultrasoundexaminations. Parameters AnnualCIMTincrement(µm/year) numberofplaquesegments sumoftheplaquethickness(mm) Patients with T2DM (n = 426) 20.33(11.74-29.86) 2.00(1.00-3.00) 5.40(2.40-7.05) Control Group (n = 137) 12.83(8.82-20.66) 1.50(0.70-2.20) 3.64(2.88-5.48) 0.50 0.00 0.00 0.02 CIMT:carotidintima-mediathickness;:variablevaluechangesduringtheobservationperiod,expressedasapercentageofbaselinevalues. Table 3.Comparisonofultrasoundmarkersofthecarotidarteryatherosclerosisinpatientswithtype2diabetesmellitus accordingtothePECAMgenepolymorphism373C/G(rs668)genotypesatthebeginningofthestudy. Parameters CIMT(µm) Numberofsegmentswithplaques Sumoftheplaquethickness(mm) Presenceofplaques:[+] [­] Presenceofunstableplaques:[+] [­] CIMT:carotidintima-mediathickness. CC Genotype 1041.00±198.00 2.56±1.65 8.14±4.77 149.00(85.60) 25.00(14.40) 86.00(57.70) 63.00(42.30) CG Genotype 990.00±187.00 2.39±1.72 7.99±5.23 256.00(84.50) 47.00(15.50) 144.00(56.30) 112.00(43.70) GG Genotype 988.00±211.00 2.51±1.54 7.66±4.02 94.00(79.70) 24.00(20.30) 58.00(61.70) 36.00(38.30) p value 0.19 0.36 0.16 0.36 0.66 Table 4.Changesinultrasoundmarkersofcarotidarteryatherosclerosisinpatientswithtype2diabetesmellitusbetween thefirstandthesecondultrasoundexaminationofthecarotidarteriesaccordingtothePECAMgenepolymorphism 373C/G(rs668)genotypes. Parameters AnnualCIMTincrement(µm/year) numberofplaquesegments sumoftheplaquethickness(mm) CC Genotype 20.34(11.64-28.04) 2.00(0.50-2.50) 7.30(3.85-8.70) CG Genotype 20.69(7.14-32.28) 2.00(1.00-3.00) 5.45(2.30-9.20) GG Genotype 14.28(10.71-35.97) 2.00(2.00-3.00) 4.40(1.40-8.42) p Value 0.71 0.74 0.38 CIMT:carotidintima-mediathickness;:variablevaluechangesduringtheobservationperiod,expressedasapercentageofbaselinevalues. weremoresmokerscomparedtothecontrolgroup. There were no statistically significant differences betweenpatientswithT2DMandcontrolsinother clinicalcharacteristics[age,bodymassindex(BMI), systolicanddiastolicpressure].AbiochemicalexaminationofpatientswithT2DMshowedstatistically significanthigherlevelsoffastingglucose,HbA1c, totalcholesterol,HDL,LDL,triglycerideandhsCRP comparedwiththecontrolgroup(Table1).Moreover, higherCIMTwasfoundinpatientswithT2DMin comparisonwithsubjectswithoutT2DM(Table1). Theultrasoundexaminationofthecarotidarterywasperformedatthetimeofenrollmentinthe study,and3.8±0.5yearsaftertheinitialexamination. Changesintheprogressionofatheroscleroticmarkers (changeinannualCIMTincrease,changeinthenumberofplaquesegmentsandchangeinthesumofthe plaquethickness)betweensubjectswithT2DMand thecontrolgroupareshowninTable2.Statistically significantlyfasterprogressionoftheatherosclerotic markerswasshowninsubjectswithT2DMincomparisonwiththecontrolgroup(Table2). Thedistributionofgenotypesinthepopulation ofpatientswithT2DMwasinHardy-Weinbergequilibrium(T2DM:2=0.45;p=0.50;controlgroup: 2=1.46;p=0.23).Table3outlinesdifferencesin theultrasoundmarkersofcarotidarteryatherosclerosis(initialultrasoundexamination)inpatientswith T2DMwithregardtothers668genotypes.Thedifferenceswerenotstatisticallysignificantwithregard tors668genotypes(Table3).Wedidnotdemonstrate statisticallysignificantdifferencesinthemarkersof subclinicalcarotidatherosclerosisbetweendifferent genotypesinsubjectswithT2DM(Table4). Table5showstherelationbetweenthers668and theincidenceofeitherplaquesorunstableplaques Table 5.RelationshipofthePECAMgenepolymorphism373C/G(rs668)genotypestothepresenceofplaques/unstable plaquesofthecarotidarteriesinpatientswithtype2diabetesmellitusatthebeginningofthestudy. Presence of Plaques Parameters Hypertension(0=no;1=yes) Systolicpressure(mmHg) SerumLDL(mmol/L) SerumHDL(mmol/L) HbA1c(%) CG allele GG allele OR (95% CI) 1.88 0.24 1.41 0.11 0.89 1.03 1.18 p Value 0.35 0.68 0.41 0.02 0.03 0.49 0.03 Presence of Unstable Plaques OR (95% CI) 1.38 0.26 1.34 0.31 1.23 0.72 0.68 p Value 0.52 0.32 0.25 0.34 0.43 0.17 0.51 OR(95%CI):oddsratio(95%confidenceinterval);LDL:low-densitylipoprotein;HDL:high-densitylipoprotien;allmodelswereadjustedbyage, sex,smokinghabitsandtreatmentwithstatins.ThereferencegrouparehomozygotesfortheCallele(theCCgenotype). insubjectswithT2DM.Whenadjustedtootherrisk factors,thers668GGgenotypewasassociatedwith anincreasedriskofcarotidplaquesinsubjectswith T2DM(Table5). DISCUSSION Inthepresentstudy,wedemonstratedanassociationbetweenthers668GGgenotypeandcarotid arteryplaqueincidenceinpatientswithT2DMwhen adjustedtootherriskfactors.Ontheotherhand,we didnotfindanyeffectofthers688genotypeonthe progressionofatherosclerosisinpatientswithT2DM. Ourstudyisthefirstdemonstratinganassociationbetweenthers668PECAM-1andthepresenceof carotidplaquesinpatientswithT2DM.Inourstudy, weobservedagreaternumberofplaquesinsubjects withtheGGrs668genotype.Theimportanceofthe PECAM-1geneinthepathogenesisofatherosclerosis wasdemonstrated[21,25].Thedecreaseinareaswith atheroscleroticlesioninPECAM-1doubleknock-out micewasreported[21,25]. Anassociationbetweenthers668PECAM-1and car-diovasculardisorderswasreportedseveraltimes, butnotinallstudies[29-33].Similarly,anassociationbetweenthers668PECAM-1geneandeitherthe increasedlevelsofsolublePECAM-1orischemic strokewasfoundintheChinesepopulation[21].An associationbetweenthers668PECAM-1geneand increasedlevelsofsolublePECAM-1wasconfirmed inthesettingofpatientswithacuteMI[28]. Similarly,Fanget al.[29]demonstratedanassociationbetweenthers668PECAM-1geneandeither increasedlevelsofsolublePECAM-1ortheseverity ofcoronaryarterydiseaseintheAsianIndianpopulationinSingapore.Reschneret al.[17]reportedan associationbetweenthePECAM-1rs668(theCC genotype)andMIinCaucasianswithT2DM.Finally, theeffectofseveralpolymorphismsofthePECAM-1 geneoncardiovascular diseasewasconfirmedby Listi et al.[30]intheNorthernItalianpopulation. Thesefindingswerenotconfirmedinthegeneral populationfromGermany,asrs688wasnotfound tobeanindependentriskfactorforcoronaryartery disease(CAD)[31]. Inarecentlypublishedmeta-analysisof15studies,including7636subjects,noassociationbetween thers668PECAM-1andcardiovasculardiseaseswas demonstrated[35].Moreover,theprogressionofsubclinicalmarkersofcarotidatherosclerosiswasstatisticallysignificantlyfasterinsubjectswithT2DM incomparisonwithsubjectswithoutT2DM.This findingisinaccordancewithexpectationsofother researchersaswell[32,33]. Ourstudyhassomelimitationsduetoitsactual design(cross-sectionaldesignattheenrollmentof subjectswithT2DMandcontrolsubjects)andrelativelysmallsamplesize(i.e.,lessthan1000subjects withT2DM),howeverthestudywasappropriately poweredtodetectthedifferencesinsubclinicalmarkersofcarotidatherosclerosisuponenrollmentaswell asduringfollow-up.Thelackoftheeffectofrs688 ontheprogressionofatherosclerosismightbedueto arathershortintervalbetweenthefirstandcontrol examinations(3.8±0.5years).Moreover,itisimpossibletoexcludetheimpactofinteractionswithother potentiallyrelevantvariablesonthedevelopmentof atherosclerosis.Thesecondlimitationofthestudy isthelackofanintermediatephenotype,i.e.,serum PECAMlevels.Wepresumethattheeffectofrs668 is viaincreasedserumPECAMlevels. Inconclusion,ourstudydemonstratedaminor effectofthers668PECAM-1onthemarkersofcarotidatherosclerosisinsubjectswithT2DM,asthe GGgenotypeofthers668PECAM-1wasassociated withahigherincidenceofcarotidplaquesinsubjects withT2DM.Withthatkindofassociationsestablishedingeneticstudies,wepresumedthatwemight predictthegeneticriskofcarotidatherosclerosisin subjectswithT2DM. ACKNOWLEDGMENTS TheauthorsthankMrs.BrinaBeskonik,B.A., JezicekBrinaBeskovniks.p.,Portoroz-Portorose, Slovenia,forrevisingtheEnglish. Declaration of Interest.Theauthorsreportno conflictsofinterest.Theauthorsaloneareresponsible forthecontentandwritingofthisarticle. 8.

Journal

Balkan Journal of Medical Genetics – de Gruyter

Published: Jun 1, 2016

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