www.jped.com.br
ORIGINAL
ARTICLE
Circulating
endothelial
progenitor
cells
in
obese
children
and
adolescents
夽
,
夽夽
António
Pires
a,∗,
Paula
Martins
a,
Artur
Paiva
b,
Ana
Margarida
Pereira
c,
Margarida
Marques
d,
Eduardo
Castela
a,
Cristina
Sena
c,
Raquel
Seic
¸a
caServiceofPediatricCardiology,HospitalPediátricodeCoimbra,CentroHospitalareUniversitáriodeCoimbra(CHUC),Coimbra,
Portugal
bInstitutoPortuguêsdoSangueeTransplantac¸ão,Coimbra,Portugal
cLaboratóriodeFisiologia,InstitutodeImagemBiomédicaeCiênciasdaVida,FaculdadedeMedicina,UniversidadedeCoimbra,
Coimbra,Portugal
dLaboratóriodeEstatística,FaculdadedeMedicina,UniversidadedeCoimbra,InstitutodeImagemBiomédicaeCiênciasdaVida,
Coimbra,Portugal
Received1October2014;accepted13January2015 Availableonline29August2015
KEYWORDS
Pediatricobesity; C-reactiveprotein; Monocyte
chemoattractant protein-1; E-selectin; Asymmetric dimethylarginine; Endothelial progenitorcells
Abstract
Objective: Thisstudy aimed toinvestigate therelationship between circulating endothelial
progenitorcellcountandendothelialactivationinapediatricpopulationwithobesity.
Methods: Observational and transversal study, including120 children andadolescents with
primary obesityofbothsexes, aged6---17 years,whowere recruitedatthis Cardiovascular
RiskClinic.Thecontrolgroupwasmadeupof41childrenandadolescentswithnormalbody
massindex.Thevariablesanalyzedwere:age,gender,bodymassindex,systolicanddiastolic
bloodpressure,high-sensitivityC-reactiveprotein,lipidprofile,leptin,adiponectin,
homeo-stasismodelassessment-insulinresistance,monocytechemoattractantprotein-1,E-selectin,
asymmetricdimethylarginineandcirculatingprogenitorendothelialcellcount.
夽 Pleasecitethisarticleas:PiresA,MartinsP,PaivaA,PereiraAM,MarquesM,CastelaE,etal.Circulatingendothelialprogenitorcellsin obesechildrenandadolescents.JPediatr(RioJ).2015;91:560---6.
夽夽StudylinkedtotheHospitalPediátrico,CentroHospitalareUniversitáriodeCoimbra(CHUC);LaboratóriodeFisiologiaandLaboratório deEstatística,InstitutodeImagemBiomédicaeCiênciasdaVida,FaculdadedeMedicina,UniversidadedeCoimbra;andtotheInstituto PortuguêsdoSangueeTransplantac¸ão,Coimbra,Portugal.
∗Correspondingauthor.
E-mail:pires1961@gmail.com(A.Pires). http://dx.doi.org/10.1016/j.jped.2015.01.011
Results: Insulinresistancewascorrelatedtoasymmetricdimethylarginine(=0.340;p=0.003),
which was directly,but weakly correlated toE-selectin (=0.252;p=0.046).High
sensitiv-ity C-reactiveprotein wasnot foundto becorrelated tomarkersofendothelial activation.
Systolicbloodpressurewasdirectlycorrelated tobody massindex(=0.471;p<0.001)and
thehomeostasismodelassessment-insulinresistance(=0.230;p=0.012),andinversely
corre-latedtoadiponectin(=−0.331;p<0.001)andhigh-densitylipoproteincholesterol(=−0.319;
p<0.001).Circulatingendothelialprogenitorcellcountwasdirectly,butweaklycorrelated,to
bodymassindex(r=0.211;p=0.016),leptin(=0.245;p=0.006),triglyceridelevels(r=0.241;
p=0.031),andE-selectin(=0.297;p=0.004).
Conclusion: Circulatingendothelialprogenitorcellcountiselevatedinobesechildrenand
ado-lescentswithevidenceofendothelialactivation,suggestingthat,duringinfancy,endothelial
repairingmechanismsarepresentinthecontextofendothelialactivation.
©2015SociedadeBrasileiradePediatria.PublishedbyElsevierEditoraLtda.Allrightsreserved.
PALAVRAS-CHAVE
Obesidadeinfantil; ProteínaCreativa; Proteínaquimiotática demonócitos-1; E-seleticna; Dimetilarginina assimétrica; Célulasprogenitoras endoteliais
Célulasprogenitorasendoteliaiscirculantesemcrianc¸aseadolescentesobesos
Resumo
Objetivo: Oobjetivodesteestudofoi investigararelac¸ãoentreosnúmerosdecélulas
pro-genitoras endoteliaiscirculantese aativac¸ãoendotelial em uma populac¸ãopediátrica com
obesidade.
Métodos: Estudoobservacionaletransversal,incluindo120crianc¸aseadolescentescom
obesi-dadeprimáriadeambosdesexos,comidadesentre6e17anos,recrutadosdenossaClínica
deRiscosCardiovasculares.Ogrupodecontrolecontoucom41crianc¸aseadolescentescom
índicedemassacorporalnormal.Asvariáveisanalisadasforam:idade,sexo,índicedemassa
corporal,pressãoarterialsistólicaediastólica,proteínaCreativadealtasensibilidade,perfil
lipídico,leptina,adiponectina,resistênciaàinsulinaparaavaliac¸ãodomodelodehomeostase,
proteínaquimiotáticademonócitos-1,E-seleticna,dimetilargininaassimétricaenúmerosde
célulasendoteliaisprogenitorascirculantes.
Resultados: Aresistênciaàinsulinafoicorrelacionadaadimetilargininaassimétrica(p=0,340;
p=0,003),que foidiretamentecorrelacionada, porém deformamuitaamena àE-seleticna
(=0,252; p=0,046). Nãoconstatamos quea proteína C reativa de alta sensibilidade está
correlacionada amarcadores de ativac¸ãoendotelial. A pressãoarterial sistólicafoi
direta-mentecorrelacionadaaoíndicedemassacorporal=0,471;p<0,001)eàresistênciaàinsulina
paraavaliac¸ãodomodelodehomeostase(=0,230;p=0,012)einversamentecorrelacionada
a adiponectina(=-0,331; p<0,001)e lipoproteína de alta densidade-colesterol =-0,319;
p<0,001).Osnúmerosdecélulasprogenitorasendoteliaiscirculantesforamdiretamente
cor-relacionados,porémdeformamuitoamenaaoíndicedemassacorporal(r=0,211;p=0,016),
àleptina(=0,245;p=0,006),aosníveisdetriglicerídeos(r=0,241;p=0,031)eàE-seleticna
=0,297;p=0,004).
Conclusão: Osnúmerosdecélulasprogenitorasendoteliaiscirculantessãoelevadosemcrianc¸as
eadolescentesobesoscomcomprovac¸ãodeativac¸ãoendotelial,sugerindoque,nainfância,os
mecanismosdereparac¸ãoendotelialestãopresentesnocontextodaativac¸ãoendotelial.
©2015SociedadeBrasileiradePediatria.PublicadoporElsevierEditoraLtda.Todososdireitos
reservados.
Introduction
Inobesity,variousinflammatoryagents,suchasC-reactive protein1---3andleptin,4disturbtheproductionofnitricoxide
via the inhibition of its rate-limitingenzyme, endothelial nitric oxide synthase (eNOS).5,6 Other endogenous
sub-stances, such as asymmetric dimethylarginine (ADMA), a competitive antagonist of eNOS, also play a role in fur-thercompromisingnitricoxidebioavailability.Itsproduction is stimulated by inflammatory agents, such as C-reactive
protein.7 Unlike nitricoxide,it canbe easilyassayed and
usedasasurrogatefornitricoxidebioavailability.
Inpatientswithcardiovascularriskfactors,suchas arte-rial hypertension and insulin resistance, the count and function of EPCs is reduced. In these, the risk of cardio-vasculareventsisincreased.9,10
Inflammatorymarkersalsoappearstoreducethenumber ofEPCs,implyingapossibleroleinobesity.11,12
The aims of the present study were two fold. Firstly, to demonstrate that endothelial activation is present in childhoodobesity;andsecondly,thatrepairingmechanisms, throughEPCactivation,arenot,atthisearlystage, compro-mised.
Methods
Subjects
The authors conducted an observational and transversal analysis, in a cohort of obese children and adolescents, recruitedrandomly,andfollowed-upataCardiovascularRisk Clinic.Allparentsgavetheirinformedconsentforthe chil-drentoparticipateinthestudy,whichhadbeenapproved bythelocalEthicsCommittee.
Theinclusioncriteriaforthestudy groupwereprimary obesity(bodymassindex[BMI]abovethe95thpercentilefor sexandage)inchildrenandadolescentsaged6---17years, withoutrecent or chronic illnesses.The exclusion criteria included secondary causes of obesity,acute infectious or inflammatorydisorders(within amonth of sampling),and chronicdisorders.
Thecontrolgroupincludedhealthychildrenand adoles-cents,unrelated tothe studygroup, withinthe sameage rangewitha normal BMI(percentil 5---85), recruited from theCardiologyClinicwheretheyweresentforevaluationof murmurs,butprovedtohavenosubjacentcardiac anoma-lies. All had undergone a 12-h fast prior to the clinical evaluationandbloodsampling.
The study group comprised 120 and the control group 41children andadolescents,of both sexes. Basedonthis samplesize,asignificancelevel(p-value)of0.05,apower of 0.80,and an effect size of 0.52were obtained. These valueswerecalculatedusingtheG*Power3.1.5program.
The variables analyzed were: age, gender, body mass index,systolicbloodpressure,diastolicbloodpressure,total cholesterol, high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides, high-sensitivity C-reactive protein (hsCRP), monocyte chemoattractant protein-1 (MCP-1), lipid profile, leptin, adiponectin, homeostasis model assessment-insulin resis-tance(HOMA-IR),E-selectin,asymmetricdimethylarginine, andcirculatingEPCcount.
Clinicalandanthropometricevaluation
Weight(inkilogramstothenearest100g)wasdetermined usingaSECA220® digitalweightscale(MedicalScalesand Measuring Systems, Hamburg, Germany) and for standing height(incentimeterstothenearest0.1cm)astadiometer includedinthesameequipmentwasused,withthechildren wearingonly undergarments. Body mass indexwas calcu-lated basedon the formula:BMI=(weight/height2).13 The
WorldHealthOrganization(WHO)BMIpercentilechartswere
usetodefineobesity(ifBMI>percentil95;normalweight: BMIbetweenpercentil5---85).
The criteria for metabolic syndrome in children and adolescentsweredefinedaccordingtotheInternational Dia-betesFederationconcensus.14
Bloodcollectionandbiochemicalanalysis
Fastingvenousbloodsamples(15mL)wereobtainedto esti-matethehematologicalparameters.Bloodspecimenswere collected in vacutainertubes withor without ethylenedi-aminetetraaceticacid(EDTA)asneeded.Serumandplasma were preparedand then frozen (−80◦C)for storage until
analysis. High-sensitivity C-reactive protein was deter-mined by immunonephelometry from serum samples and processedintheBNProSpec® System(SiemensHealthcare DiagnosticsInc,Munich,Germany)analyzer(undetectedif <0.02mg/dL).
Monocyte chemoattractant protein-1, asymmetric dimethylarginineandE-selectinweredeterminedbyELISA using commercially available enzyme-linked immunosor-bent assaykits OptEIATM (BD BiosciencesPharmingen, CA, USA).
Leptin and adiponectin levels were determined using commerciallyavailableenzyme-linkedimmunosorbentassay kits(eBioscience--- SanDiego,CA,UnitedStatesand BioVen-dor--- Brno,CzechRepublic,respectively).
Insulin levels were determined by chemiluminescence fromserumsamplesandprocessedintheIMMULITE2000® (SiemensHealthcareDiagnosticsInc,Munich,Germany) ana-lyzer and glucose levels were determined from plasma samplesanalyzedintheVITROS5.1FS®(OrthoClinical Diag-nostics,Johnson&Johnson,NY,USA)systembymicroslide technology.
The HOMA-IR was calculated based on the formula; HOMA-IR=insulin(mU/L)×glucose(mmol/L)/22.5, consid-ering 3 as the cut-off value for the diagnosis of insulin resistance.15
Total cholesterol, low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol (HDL-C),and triglyceride (TG) concentrations were measured using an automatedbiochemicalanalyzerVITROS5.1FS®(Ortho
Clin-icalDiagnostics,Johnson&Johnson,NY,USA).
Fordeterminationof circulating EPC count,peripheral blood (PB) was collected in EDTA tubes, stored on ice, and processed within2h. Identification and characteriza-tionofcirculatingEPCwasperformedusingananti-CD146 conjugated withflouresceinisothiocyanate(clone:P1H12, BectonDickinson(BD),CA,USA),anti-KDRconjugatedwith phycoeritrin (clone: 89106, R&D System, Headquarters, Minneapolis, USA), anti-CD34 peridinin chlorophyll pro-tein cyanine 5.5 (clone: 8G12, BD Bioscience, CA, USA), anti-CD133allophycocyanin(clone:293C3,MiltenyiBiotec, Bergisch Gladbach, Germany), and CD45 krome orange (clone: J.33, Immunotech --- Beckman Coulter, Marseille, France)combinationofmonoclonalantibodies(mAb).
Forsamplestaining,adirectimmunofluorescence tech-nique was used. Data acquisition was performed in a FACSCantoII® flowcytometer(BDBiosciencesPharmingen,
CA, USA) using the FACSDiva® software (BD Biosciences
International Society for Hematotherapy and Graft Engi-neering (ISHAGE) sequential gating strategy proposed by Schmidt---Luckewasfollowed.
CirculatingEPCSwereidentifiedaccordingtoaminimal antigenicprofilethatincludesatleastonemarkerof stem-ness/immaturity(CD34andCD133),plusatleastonemarker ofendothelialcommitment(KDRandCD146).CD45staining wasusedtoexcludeleukocytes.
Fordataanalysis,theInfinicytTMsoftware,V.1.5
(Cytog-nosSL,Salamanca,Spain)wasused.
Statisticalanalysis
ThedatawasanalyzedusingtheIBMSPSS20software(IBM Corp.Released 2011.IBM SPSSStatisticsfor Windows,NY, USA).The descriptiveanalysisof theparametric variables wasdone bycalculating themean±standard error of the mean.Student’st-testandMann---WhitneyUtestwereused tocalculate thedifferences in the demographic,clinical, andhematologicalparametersbetweentheobeseand con-trolgroups,dependingonthenormalityofdistribution.For categoricalresponsevariables,differencesbetweenthetwo groups were assessed using the chi-squared test. Logistic regression was used when clinical variables were con-trolledbetweenthetwogroups.Toestablishthecorrelation betweentheparametersintheobesegroup,Spearman’sand Pearsons’scorrelationswereused.Theresultswere consid-eredstatisticallysignificantatp<0.05.
Results
Comparativeanalysisbetweentheobeseand controlgroups
Onehundredandtwentyobesechildrenandadolescents,61 boysand59girls,withagesbetween6and17years(mean age11.65years±2.96)wereincludedinthestudy.The con-trolgroup wasmade upof41 healthy,non-obesechildren andadolescents,29boysand12girls,withinthesameage group(meanage12.73years±2.77).
We firstly compared the two group’s anthropometric, clinical and analytical parameters. As demonstrated in
Table1,allthemeansoftheanalyzedparameterswere sig-nificantlyhigher in the obese group,including E-selectin, ADMAandcirculatingEPCS,exceptforadiponectinand HDL-C(Fig.1).Totalcholesterolandmonocytechemoattractant protein-1showednosignificantstatisticaldifferences.
As age (p=0.038) and gender (p=0.027) were signif-icantly different between the two groups, the analysis wasrepeatedusinglogisticregression,andtheresultsdid not differ significantly, namely, MCP-1 (p=0.334), ADMA (p=0.005),E-selectin(p=0.008),andEPCs(p=0.043).Total cholesterolandMCP-1continuedtoshownosignificant dif-ferences.
Circulating EPCcount were directly,but weakly corre-lated,toBMI(r=0.21;p=0.016),leptin(=0.25;p=0.006),
triglyceride levels (r=0.24; p=0.031), and E-selectin (=0.30;p=0.004).
Table1 Anthropometric,clinical,andanalyticalparametersoftheobeseandnon-obesegroups.
Variable Obese Non-obese p-value
n Mean±SEM n Mean±SEM
Age(years) 120 11.65±0.43 41 12.73±0.27 0.038a
BMI(kg/m2) 120 28.47±0.44 41 18.93±0.43 <0.001b
sBP(mmHg) 120 116.60±1.16 41 107.77±1.75 <0.001b
dBP(mmHg) 120 61.19±0.69 41 54.95±1.41 <0.001a
Leptin(ng/mL) 118 28.97±1.63 41 4.85±0.71 <0.001a
Adiponectin(g/mL) 95 3.60±0.14 32 5.17±0.41 <0.001a
Insulin(UI/mL) 119 12.95±0.73 33 7.32±0.75 <0.001a
HOMA-IR 118 2.81±0.17 33 1.54±0.15 <0.001a
MCP-1(pg/mL) 65 331.54±46.49 31 304.53±48.33 NS
hsCRP(mg/dL) 118 0.2781±0.03 40 0.05±0.09 <0.001a
CT(mg/dL) 120 166.86±2.52 41 163.83±5.39 NS
HDL-C(mg/dL) 120 47.33±0.90 41 59.17±2.04 <0.001a
LDL-C(mg/dL) 120 96.45±2.44 41 82.71±3.74 0.004b
TG(mg/dL) 120 82.71±4.93 41 59.15±4.28 <0.001a
ADMA(mol/L) 76 0.44±0.01 31 0.38±0.02 0.005a
E-selectin(pg/mL) 77 12.8±0.84 30 9.03±0.92 0.003b
EPCs(numbers) 100 1,565,532.31±56,764.76 29 1,328,311.00±64,449.43 0.007b
Malen(%) 120 61(50.8%) 41 29(70.7%) 0.027b
BMI, bodymassindex; sBP, systolicbloodpressure; dBP,diastolic blood pressure;HOMA-IR, homeostasismodel assessment-insulin resistance;MCP-1,monocytechemoattractantprotein-1;hsCRP,high-sensitivityC-reactiveprotein;CT,totalcholesterol;HDL-C,high densitylipoproteincholesterol;LDL-C,lowdensitylipoproteincholesterol;TG,triglycerides;ADMA,asymmetricdimethylarginine;EPCs, circulatingendothelialprogenitorcellcount;NS,non-significantp-value;n,samplenumber;SEM,standarderrorofthemean.
Obese 0
500,000 1,000,000
Mean endothelial progenitor cell numbers
1,500,000 2,000,000
*
Non-obese
Figure1 Endothelialprogenitorcellcountintheobeseand
thenon-obesegroups.Thehigherlevelsintheobesegroupmay
reflecttheresponsebyrepairingmechanismssecondarytoearly
endothelial activation. Data expressed as means±standard
error of the mean. Obese group, n=100; non-obese group,
n=29.*Student’st-test,p<0.01vs.nonobesegroup.
Analysisoftheobesegroup
Bodymassindexwasdirectlyandmoderatelycorrelatedto MCP-1(=0.51;p<0.001),systolicbloodpressure(=0.47;
p<0.001),leptin(=0.40;p<0.001),andHOMA-IR(=0.35;
p<0.001);weaklycorrelatedtohsCRP(=0.22;p=0.018); and inversely but weakly correlated to adiponectin (=−0.28;p=0.007)andHDL-C(=−0.26;p=0.004).
Monocyte chemoattractant protein-1 was directly cor-related tosystolic blood pressure (=0.34; p=0.005), as shown in Fig. 2. High sensitivity C-reactive protein was
90.00 .000 500,000 1,000,000 1,500,000 2,000,000
R2 linear=0.099
100.00 110.00 120.00
sBP
MCP-1
130.00 140.00 150.00
Figure 2 Correlation between monocyte chemoattractant
protein-1andsystolicbloodpressure.Inobesity,cardiovascular
riskfactorsclusterandcontributetotheinflammatoryprocess,
asobservedinthecorrelationbetweensystolicbloodpressure
andMCP-1inthe presentstudy.sBP,systolic bloodpressure;
MCP-1,monocytechemoattractantprotein-1.
weaklycorrelatedtoleptin (=0.32;p<0.001) andLDL-C (=0.23;p=0.011).
Systolic blood pressure was inversely correlated to adiponectin (=−0.33; p=0.001) and directly correlated toHOMA-IR (=0.23;p=0.012) and,asexpected, moder-atelycorrelatedtoage(=0.50;p<0.001).Diastolicblood pressurehadlesspronouncedcorrelations.
TheHOMA-IRwascorrelatedtoADMA(=0.34;p=0.003) providing the most direct link to a marker of endothe-lial activation. This was evident particularly at HOMA-IR values above 3 (p=0.003), the cut-off value we used to define insulin resistance. It was also directly cor-related to leptin (=0.39; p<0.001), systolic blood pressure (=0.23;p<0.012), anddiastolic blood pressure (=0.20;p<0.033),andinverselycorrelatedtoadiponectin (r=−0.21;p=0.040).
Metabolic syndrome was observed in 11.7% (n=14) of the obese patients. As expected, being part of the definition,systolic(p=0.005),butnotdiastolicblood pres-sure (p=0.728), waselevated, aswere triglyceridelevels (p<0.001),HDL-Cwaslower(p=0.007),andnosignificant differenceswereobservedinglucoselevels.Regardingthe markers of endothelial activation andrepair mechanisms, ADMA was directly, but weakly correlated to E-selectin (=0.25; p=0.046),and thelatter wasweaklycorrelated tocirculatingEPCcount(=0.22;p=0.047).
Discussion
The present study clearly demonstrates that, unlike their lean counterparts, obese children and adolescents with evidence of ongoing low-grade inflammation and endothelialactivation haveraised EPCcount.Correlations between adiposity, inflammatory markers, and obesity-related comorbidities such as arterial hypertension and insulinresistancewerealsoobserved.
Monocytechemoattractantprotein-1regulatesmigration and infiltration of monocytes andmacrophages16 into the
vascularwallandplaysanimportantrolein inflammation-mediateddiseases,suchasobesity.17 Asscanttranslational
researchhasbeendoneinthisfield,theauthorswere inter-ested in evaluating the relationship of MCP-1 to obesity relatedcomorbidities.UnlikethefindingsbyBreslinetal.,18
nodifferenceswereobservedinMCP-1levelsbetweenthe obeseandcontrolgroupsnorwhatanassociationwith dys-lipidemia retrieved (data not shown), possibly due to a different sample size(39 obesechildren andadolescents) and ethnicity (American-Mexican). However, in the obese group, MCP-1,unlike hsCRP, hada directcorrelation with systolic blood pressure. This trend was not found in the controlgroup.Itis,thustemptingtopostulatethatin child-hood obesity,MCP-1 contributes to arterial hypertension, andimplicitlyisimplicatedinthepathophysiological mech-anismsleadingtocardiovasculardiseaselaterinlife.Based onthepresentevidence,MCP-1,comparedtohsCRP,would appeartobeamorerobustcardiovascularriskmarker. How-ever,duetothesamplesize,furtherresearchneedstobe done in thisfield.To thebestof theauthors’ knowledge, theseassociationshavenotbeenpreviouslydescribed.
highersystolicanddiastolicbloodpressurevalues(p<0.001) than thecontrol group.The prevalence of arterial hyper-tension in the obese group was9.2%, and almost a third had pre-hypertension (26.7%). Apart from MCP-1, and in agreementwiththefindingsbyMoseretal.,20systolicblood
pressurewasdirectlycorrelatedtoBMIandinverselyrelated to adiponectin and HDL-C. This cluster of mediators cor-relatesvisceraladiposity,inflammation,insulinresistance, andhypertension,someofthecomponentsofthemetabolic syndrome, found in 11.7% of the obese group. A particu-larinterestingfindingwastheinversecorrelationbetween adiponectin and arterial hypertension, thus corroborating the findings of the study by Brambilla et al.,21 in which
adiponectinwasproposedasoneofthemechanismsrelated tohypertensioninchildhoodobesity.This impliesthatthe lossofitsantiatherogenicandanti-inflammatoryproperties contributetotheunderlyingmechanismsofobesity-related hypertension.
Furthermore,thelossofadiponectin’sinsulinsensitizing properties that occurs in obesity has also been impli-cated in insulin resistance.22 Unlike the findings by Lee
etal.,23 whereadiponectinwasfoundtobeastrong
inde-pendent predictor of insulin sensitivity in obese children and adolescents of different racial backgrounds, in the present study adiponectinwas shown to becorrelated to hyperinsulinaemia(r=−0.23; p=0.033),butnot toinsulin resistance (n=38%; p=0.277), despite strikingdifferences inadiponectinlevelsbetweentheobeseandcontrolgroups (p<0.001).
The present study included only whitesubjectsfrom a differentgeographicalarea,which,togetherwiththe sam-ple size (n=45 with HOMA-IR >3), might account for the observeddifferences,although adiponectingene polymor-phisms might also play a role. This hypothesis is merely speculative,asitwasnottestedinthepresentstudy. How-ever, this rationale could not beapplied toleptin, which had a stronger correlation to hyperinsulinaemia (r=0.35;
p<0.001) and insulin resistance (p<0.001), findings in accordancewithpreviouslypublishedworks.24Aconnection
between MCP-1 (p=0.168), hsCRP (p=0.375), and insulin resistancewasnotobserved.
The authors further attempted to relate ADMA and E-selectin,bothmarkersofendothelialactivation,toinsulin resistance.Inadults,raisedlevelsofasymmetric dimethy-larginineareconsideredacardiovascularriskmarker,andas asurrogateofnitricoxidebioavailability,reflects endothe-lial integrity. A direct correlation between HOMA-IR and ADMA (r=0.35; p=0.003) was observed, particularly evi-dent intheinsulin-resistant group(p=0.003),findings not previously reported. Some reports have suggested that ADMAcontributestoinsulinresistance asdecreasednitric oxide bioavailability reduces blood flow to insulin sensi-tive tissues compromising glucose uptake in response to insulin.25Conversely,insulinresistanceitselfhasbeenfound
tocontributetowardendothelial dysfunction.Underthese circumstances, itis temptingtospeculate onADMA’s per-missive role on the interplay between insulin resistance and endothelial dysfunction, through its actions onnitric oxide bioavailability. A direct correlation between leptin andADMAwasalsoobserved,butonly ingirls. Thisis not surprising,asleptinlevelsweresignificantlyhigherinobese girls (p<0.001). Nevertheless, this finding, not previously
reported,potentially links leptin, and henceadiposity, to endothelialactivation.Comparatively,linearregressionby logarithmic transformation showed that insulin resistance hadagreaterinfluenceonADMA.
E-selectin is a specific endothelial adhesion molecule whoselevelsareraisedinendothelialdysfunction, promot-ingthemigrationofinflammatorycellstotheintima.Inthis analysis,obese children andadolescents had significantly higherlevelsthanhealthycontrols, implyingthepresence ofendothelialactivation,whichindicatesanearlystageof atherosclerosis.Consideringtheentirecohort,adirect,but weakcorrelation tohsCRP (=0.21;p=0.033) wasfound,
reinforcing the role of inflammation in endothelial dys-functionandinagreementwithpublishedliterature,26 but
asimilar pattern wasnot demonstrated withinthe obese group,possiblydue toitshomogeneity.Wealsoobserved, withintheobesegroup,adirectcorrelationbetweenADMA and circulating EPC count. We found these correlations particularly interesting as they highlight various patho-physiological pathways related to endothelial activation, namely,inflammatorycelladhesiontriggeredbyE-selectin, increasedexpression of ADMA, whocompetitively inhibits eNOS,reducingnitricoxidebioavailabilityand,ultimately, repairofinjuredtissueby EPCS.The authorsbelievethat therelationshipbetweenE-selectinandADMA,inchildhood obesity,hasnotbeenpreviouslypublished.
Theterm EPCsshouldbereservedforaprogenitorcell restrictedspecifically totheendothelial lineage.In 1997, Asahara et al.27 reported for the first time on the
exist-enceof EPCs.To date,nospecific markerhasbeen found toclearlyidentifythesecells,whichmakestheirisolation controversial,astheiridentificationisbasedoncellsurface markersshared byother hematological celllines.As such variousmethodshavebeen usedtoidentifyEPCs.Inorder tominimizefalsepositivesvariouscellsurfacemarkershave beenintegratedtoidentifythesecells,namelythoselinked toimmaturecelllines(e.g.CD34)withendothelial commit-ment(e.g.kinaseinsertdomainreceptor).
Asreportedby variousauthors,thecountandfunction ofcirculatingEPCSnotonlycorrelateinverselywith cardio-vascularriskfactors,butalsomaypredicttheoccurrenceof cardiovascularevents,particularlyintheadultpopulation.28
Data regarding EPCs and childhood obesity is scarce. A reportbyJungetal.29observedadirectcorrelationto
E-selectin.Contrarytothepresentstudy,thiscorrelationonly appliedtoobeseadolescents.Inthepresentstudy,EPCsand E-selectin were directly correlated in the pre-adolescent obesegroup,afindingnotpreviouslyreportedandthat rein-forcesthefactthatendothelialdysfunctionispresentinvery youngindividuals.
Inthisanalysis,theobesegrouphadasignificantlyhigher numberofEPCs,implyingthatelevatedEPCsinobese chil-dren and adolescents represent an attempt at repairing underlyingvascular damage. Thus,at this stage,it would bepossibletoreversetheunderlyingendothelialdamageif stepsaretakentocontrolweight.
damage. The inference is that therapeutic interventions aimedatweightlossoughttobeaggressivelyinstitutedin order to reverse endothelial damage and obesity-related comorbidities, such as arterial hypertension and insulin resistance.
Thisstudyhasseverallimitations.Firstlyitssamplesize, particularlyitscontrolgroup.Italsoonlyincludeda white-onlypopulation,limitedtoaparticulargeographicalarea, theformerduetolocalracedistribution.Thestudyincluded bothgenders,aswellas,differentagegroups.Intheresults, these differences were adjusted for, and therefore their interferencewasnullified.
Conflict
of
interest
Theauthorsdeclarenoconflictsofinterest.
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