Type1DiabetesIncreasestheExpressionof Proinflammatory Cytokines andAdhesionMolecules in the ArteryWallof Candidate Patients for KidneyTransplantationJAVIER TRIÑANES, MSC

1

EDUARDO SALIDO, MD, PHD1

JULIÁN FERNÁNDEZ, MD, PHD2

MARGARITA RUFINO, MD, PHD3

JOSÉ MANUEL GONZÁLEZ-POSADA, MD, PHD3

ARMANDO TORRES, MD, PHD3

DOMINGO HERNÁNDEZ, MD, PHD4

OBJECTIVEdDiabetes may accelerate atheromatosis in uremic patients. Our aim was toassess the influence of type 1 diabetes on the atheromatosis-related inflammation in patientswith chronic kidney disease (CKD).

RESEARCH DESIGN AND METHODSdWe analyzed the expression of proinflamma-tory cytokines and adhesion molecules in the inferior epigastric artery walls of type 1 diabeticpatients with CKD (n = 22) and compared it with nondiabetic uremic patients (n = 92) at the timeof kidney transplantation.We evaluated the expression of interleukin (IL)-6,monocyte chemotractantprotein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule-1,and the activation of nuclear factor-kb p65 (NFkB-p65). Common carotid intima-media thick-ness (c-IMT) was determined by conventional echography.

RESULTSdIL-6, MCP-1, and VCAM-1 proteins were elevated in type 1 diabetic patientscompared with nondiabetic subjects (P , 0.05). The nuclear localization of NFkB-p65 washigher in type 1 diabetic patients (P , 0.01) and correlated with the levels of MCP-1 in thisgroup (r = 0.726, P, 0.001). Arterial fibrosis correlated with IL-6 and MCP-1 levels (r = 0.411,P , 0.001 and r = 0.378, P = 0.001). A significant correlation was observed between VCAM-1levels and both the degree of arterial narrowing and c-IMT.

CONCLUSIONSdType 1 diabetes produces a proinflammatory state in the arteries of end-stage CKD patients, with increased levels of IL-6, MCP-1, and VCAM-1, as well as a greater degreeof p65 activation, which are associated with more severe vascular lesions and higher c-IMT.Although causality is not demonstrated, these findings support the major role of inflammation intype 1 diabetic patients with CKD.

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Vascular inflammation is an impor-tant process in the development ofcardiovascular disease (CVD), andpa-

tients with chronic kidney disease (CKD)represent a subset with a higher risk ofCVD (1,2), especially when they also havetype 1 diabetes (3). Multiple risk factors

inherent to renal failure and diabetesstatus involved in the pathogenesis ofatheromatosis concur in these patients.

Atheromatosis is an inflammatory dis-ease of the arterial wall mediated by a com-plex interaction between mononuclearcells, endothelial cells, vascular smooth

muscle cells (SMCs), growth factors, andcytokines. Indeed, the endothelium over-lying atherosclerotic lesions expresses vas-cular cell adhesion molecule (VCAM)-1and intercellular adhesion molecule(ICAM)-1, which have been shown tobe closely correlated with monocyte/macrophage infiltration and progress ofthe atherosclerotic lesion. The monocytechemoattractant protein (MCP)-1 and in-terleukin (IL)-6 also are key cytokines inthis process. In particular, MCP-1 has acrucial role in the attraction, migration,and activation of monocytes (4) and is animportant regulator for cell proliferation(5). IL-6 is a central cytokine in the inflam-matory process (6), being regulated byhigh glucose levels (7). This moleculecan also activate the expression of differentgenes, such as MCP-1, contributing tomaintaining the inflammatory milieu inthe lesion. Finally, the upregulation ofthese molecules is controlled by transcrip-tion factors, such as nuclear factor-kb(NFkB) (8) acting as a mediator in the for-mation of atherosclerotic plaques.

Experimental and clinical studieshave demonstrated the significant role ofinflammatory molecules in the setting ofatheromatosis related to type 2 diabetes(8–10). However, the expression of thesemolecules and the pathways involved intheir pathogenesis barely have been as-sessed in human tissue, especially intype 1 diabetic patients with CKD.

Common carotid intima-media thick-ness (c-IMT) is an early marker of athero-sclerosis both in the general populationand in uremic patients, including kidneytransplant recipients (11,12). Moreover,an increase in c-IMT has been observedin nondiabetic patients with prediabetesglucose alterations after kidney transplan-tation (13), but its relationship with theexpression of proinflammatory moleculesin the arterial wall in vivo has not beeninvestigated.

The purpose of this study was to assessthe influence of type 1 diabetes on the

c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c

From the 1Research Unit, University Hospital of the Canary Islands, Tenerife, Spain; the 2RadiologyDepartment,University Hospital of the Canary Islands, Tenerife, Spain; the 3Nephrology Department, University Hospitalof the Canary Islands, Tenerife, Spain; and the 4Nephrology Department, University Hospital Carlos Haya,Málaga, Spain.

Corresponding author: Domingo Hernández, domingohernandez@gmail.com.Received 29 August 2011 and accepted 8 November 2011.DOI: 10.2337/dc11-1665This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/suppl/doi:10

.2337/dc11-1665/-/DC1.© 2012 by the American Diabetes Association. Readers may use this article as long as the work is properly

cited, the use is educational and not for profit, and thework is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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atheromatosis-related inflammatory statein CKD patients at the time of kidney trans-plantation. We analyzed, in vivo, the ex-pression levels of atheromatosis-associatedinflammatory markers, such as cytokinesand adhesion molecules, and comparedthem with the levels in nondiabetic CKDpatients. We also investigated the relation-ship between subclinical atheromatosis,evaluated by c-IMT measurement, and themolecular expression of these inflammatorymarkers.

RESEARCH DESIGN ANDMETHODS

Patients and tissue samplesThis cross-sectional observational studyinitially involved 148 consecutive adultCKD patients (aged $18 years) whoreceived a single deceased kidney trans-plant or a simultaneous pancreas-kidneytransplant (n = 4) between August 2007and April 2009 at a regional transplantcenter (Hospital Universitario de Canarias,Tenerife, Spain). For the purpose of thestudy, we excluded 34 patients with type2 diabetes. Thus, the final study popula-tion comprised 114 CKD patients. Wecompared type 1 diabetic patients (n = 22)with nondiabetic recipients (n = 92). Di-agnosis of type 1 diabetes was establishedaccording to the guidelines of the AmericanDiabetes Association (14), and C-peptideplasma concentrations were determinedby immunometric assay (Immulite 2000C-Pepetide; Diagnostic Products, Los Angeles,CA). Thus, insulin-dependent individualswith low (,0.9 ng/mL) or undetectablelevels of plasma C-peptide were consid-ered to be type 1 diabetic patients. Thestudy was purely descriptive, and no at-tempts were undertaken to modify any as-pect of therapy. Also, the study wasapproved by the ethics committee of theHospital Universitario de Canarias andwas conducted according to the principlesdescribed in the Declaration of Helsinki.Each patient gave written informed con-sent to participate in the study.

During surgery, a sample of the in-ferior epigastric artery (IEA) was obtainedfrom all eligible patients. The tissue wasquickly dissected in three sections for thedifferent analyses: gene expression, proteinquantification, and histological analysis.

Histological, immunohistochemical,and immunofluorescence analysesArtery segments were fixed and paraffinembedded. Serial sectionswere cut at 3-mmthickness, and samples were stained with

hematoxylin-eosin tomeasure the propor-tion of arterial lumen reduction and withSirius Red to determine the degree offibrosis. The reduction in arterial lumenwas calculated as the percentage of lumenlost from the total lumen area measured inthe inner elastic lamina, and fibrosis wascalculated as the proportion of the total area,including media and intima layers, positivefor Sirius Red stain. We also developed aVon Kossa stain to determine the degreeof calcium deposits in the artery wall andclassified the arteries in four groups accord-ing to the severity of calcium deposits.

For immunohistochemistry, serial sec-tions were mounted on silanized slides, andafter deparaffination and heat-mediated an-tigen retrieval, the tissuewas permeabilized,blocked, and incubated with the primaryantibody overnight. The antibodies usedwere rabbit polyclonal to MCP-1 (Abcam,Cambridge, U.K.), rabbit monoclonal toICAM-1 (Abcam), and mouse monoclonalto IL-6 and VCAM-1 (Santa Cruz Biotech,Santa Cruz, CA). The immunoreactivity ofthe primary antibody was revealed by usinga peroxidase-linked antibody againstmouse/rabbit IgG and39-diaminobenzidineas a chromogen substrate.

All images were takenwith anOlympusDX41microscope (Olympus, Tokyo, Japan)fitted with a Canon DP72 camera (Canon,Tokyo, Japan) and then analyzed using thesoftware ImageJ (National Institutes ofHealth) and the Wright Cell Imaging Fa-cility (WCIF) plug-in from the WesternResearch Institute (Toronto, Canada).

For immunofluorescence, we used arabbit antibody to NFkB-p65 (Abcam),Alexa-conjugated anti-rabbit IgG as thesecondary antibody, and DAPI to stainthe nuclei. The images were taken usinga Leica confocal microscope (Wetzlar,Germany) and analyzed using the ImageJsoftware and the procedure developed byCarmona et al. (15) to measure nuclearlocalization.

RNA extractionThe sample of artery used for RNA iso-lation was quickly frozen in buffer Dcontaining guanidium thiocyanate. TotalRNA was isolated by the Chomczynskimethod (16). The purity and concentra-tion of RNA was determined by Nano-drop 2000 (Thermo-Fisher, Boston,MA).

Real-time PCRThe quantification of relative mRNA abun-dance was carried out using quantitativePCR and the SYBR green detectionmethod.

Total RNA was reverse transcribed usinga kit (Promega, Madison, WI), and thegenes of interest were PCR amplified usinggene-specific primers. Theoligonucleotidesused are shown in Supplementary Table 1.The resulting increase in fluorescence dur-ing the PCR reaction was detected in theiQ5 system (Bio-Rad, Hercules, CA), andexpression of each gene was normalizedwith the reference genes (18S rRNA andRNase P). The data were analyzed usingqBASE software (17).

Protein extractionThe sample of artery used for proteinextraction was introduced in radioimmu-noprecipitation assay buffer and immedi-ately frozen. For protein extraction, thesample was mechanically homogenized,and after centrifugation the supernatantwascollected and total protein concentrationwas determined using the bicinchoninicacidmethod (Sigma-Aldrich, St. Louis,MO).Quantification of proteins (IL-6, MCP-1,ICAM-1, and VCAM-1) was determinedusing xMAP technology (R&D Systems,Minneapolis, MN) and the Luminexplatform (Luminex, Austin, TX). Theconcentration of each analyte was deter-mined for each sample and then correc-ted by the total protein concentration;the result obtained is expressed as pico-grams of analyte per microgram of totalprotein.

c-IMT measurementsAfter surgery and within the following2 weeks, c-IMT was determined by anexperienced radiologist with no knowl-edge of the patient’s clinical data. Mea-surements were made according to thestandard clinical procedure (13) using anSSA-380 ultrasound transducer (Toshiba,Tokyo, Japan). Either an L6-7 MHz or alinear array transducer L11-7.5 MHzprobe (PLM 703AT) was used dependingon the depth of the artery. A low intraob-server variability has previously beenreported in our radiology section aftertwo repeated measurements (mean intra-class concordance correlation 0.96 [95%CI 0.90 – 0.99]; P , 0.001) (13).

Statistical analysisDescriptive data are expressed asmeans6SD. Comparisons of continuous variablesbetween the two groups were made bymeans of the Mann-Whitney U test. Thex2 test and Fisher exact test, when appro-priate, were used for between-group ana-lyses of categorical variables. The Spearmancorrelation coefficient was used to analyze

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unadjusted relationships between contin-uous data. All data were analyzed usingthe SPSS statistical package 13.0 (SPSS,Chicago, IL). A P value,0.05 was consid-ered significant.

RESULTSdNo significant differenceswere found between the two groups inmale-to-female ratio, age, BMI, type ortime on dialysis, CVD pretransplantation,total cholesterol, HDL cholesterol, trigly-cerides, smoking status, or use of statins,aspirin, and ACE inhibitors or angioten-sin II receptor blockers (ARBs) (Table 1).As expected, the main cause of renal fail-ure in the type 1 diabetic group was dia-betic nephropathy. A high prevalence ofpolycystic kidney disease also was ob-served in the nondiabetic population. Inaddition, glucose levels and glycosylatedhemoglobin (HbA1c) were significantlyhigher in type 1 diabetic patients com-pared with nondiabetic subjects.

Pathological changes of theartery wallThe distribution of the subjects in fourclasses according to the luminal narrow-ing (Supplementary Table 2) was agedependent in nondiabetic subjects (r =0.332, P = 0.003) but not in type 1 di-abetic subjects. Overall histopathologicanalysis showed a greater degree of lu-men reduction in the epigastric arteryfrom type 1 diabetic patients comparedwith nondiabetic subjects (17.9 6 19.5vs. 6.39 6 8.8%; P = 0.014) as well as agreater proportion of fibrosis in the intima(0.646 0.12 vs. 0.496 0.18; P, 0.001).

Histological and immunohistochem-ical analyses also showed that the intimathickening for the IEA was composed, atall the stages observed, of smooth muscleactin (SMA)-positive cells and collagenfibers (Fig. 1A and B), whereas CD68-positive macrophages only were presentas isolated cells in the intima and adventitia

or forming aggregates around medial cal-cifications in themost advanced stages butnever as a major cell type in the intima(Fig. 1C and D).

We classified medial calcification infour stages (absence, initial, medium, andadvanced) in all subjects. No intimal cal-cification was detected in any sample,whereas medial calcification was presentin the arteries of both groups, with ahigher incidence and severity in the type 1diabetic group (Supplementary Fig. 1).Calcification in the media layer was pres-ent in 22.9% of nondiabetic subjects andin 86.4% of type 1 diabetic patients (P ,0.001) (Supplementary Fig. 1). The varia-bles significantly associated with the stageof medial calcification in nondiabetic sub-jects were time on dialysis (r = 0.213, P =0.05) and age (r = 0.416, P , 0.001). Onthe other hand, fasting glucose levels (r =0.469, P = 0.037) seemed to be the mostrelevant factor in type 1 diabetes. Immuno-histochemical analysis showed no relation-ship between the expression of moleculesunder study in the intima and media layercalcification.

Expression of proinflammatorycytokines and adhesion moleculesin the artery wall at the time oftransplantationFigure 2A shows the gene expression inthe artery wall of proinflammatory cyto-kines and adhesion molecules in the twogroups of patients. A significantly higherexpression of MCP-1 and IL-6 was obser-ved in type 1 diabetic patients comparedwith the nondiabetic group. In addition, atrend toward higher mRNA levels fromthe adhesion molecules (ICAM-1 andVCAM-1) also was seen in type 1 diabeticpatients.

As a result, significant differences be-tween both groups also were observed forprotein quantification of IL-6, MCP-1,and VCAM-1 but not for ICAM-1 (Fig.2B). Moreover, a significant correlationbetween mRNA expression and proteinlevels was evidenced for IL-6 (r = 0.263,P = 0.003) and MCP-1 (r = 0.238, P =0.008) but not for the adhesion mole-cules, indicating that posttranscriptionalprocesses could be responsible for the dif-ferences between themRNA expression ofadhesion molecules and protein levels.

Of note, NFkB-p65 nuclear localiza-tion measured along the intima-mediawas significantly higher in type 1 diabeticpatients compared with nondiabetic sub-jects (P = 0.019) (Fig. 3) and correlatedwith the MCP-1 protein levels in the type

Table 1dClinical characteristics of both groups

Type 1 diabeticpatients

Nondiabeticsubjects P

n 22 92Sex (male/female) 15/7 69/23 0.619Age (years) 50.32 6 12.15 46.47 6 14.29 0.167BMI (kg/m2) 25.83 6 5.25 24.31 6 4.09 0.141Dialysis type (% hemodialysis) 77.3 81.5 0.743Time on dialysis (months) 29.38 6 38.47 26.61 6 27.57 0.705Pretransplant CVD (%) 22.7 10.9 0.124Ischemic heart disease 8.7 7.6Peripheral arterial disease 4.3 1.1Transient ischemic attack 9.7 2.2

Use of ACE inhibitors/ARBs (%) 11.0 8.7 0.751Use of statins (%) 17.4 15.4 0.816Use of aspirin (%) 31.8 23.9 0.533Hypertension (%) 95.5 92.4 0.579HDL (mg/dL) 39.23 6 14.88 42.9 6 12.12 0.353Total cholesterol (mg/dL) 133.14 6 36.33 136.15 6 33.39 0.592Triglycerides (mg/dL) 153.52 6 59.55 139.55 6 72.51 0.481Smokers (%) 0.256No 59.1 72.8Former 27.3 14.1Current 13.6 13.1

Cause of CKD (%) ,0.001Diabetes 81.8 dGlomerulonephritis d 21.7Polycystic kidney disease 4.5 21.7Hypertension d 4.4Nephroangiosclerosis d 8.7Other 13.6 43.5

Fasting glucose (mg/dL) 138.15 6 33.36 100.12 6 14.32 ,0.001HbA1c (%) 6.97 6 0.62 5.31 6 0.99 ,0.001

Data are means 6 SD and proportions, unless otherwise indicated. Ischemic heart disease includes myo-cardial infarction, coronary artery bypass, and stent.

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1 diabetic group (r = 0.797, P = 0.001) butnot in nondiabetic subjects.

Finally, the degree of lumen reduc-tion in the epigastric artery correlatedwith the protein levels of MCP-1 andVCAM-1 (r = 0.275, P , 0.05 and r =0.258, P , 0.05, respectively), and italso was associated to abundant SMA-positive cells in the neointima (Fig. 1EandG), whereas the degree of fibrosis cor-related with the protein levels of IL-6 andMCP-1 (r = 0.41, P, 0.01 and r = 0.378,P , 0.01, respectively) produced also by

the SMA-positive cells in both intima andmedia layers (Fig. 1E and F).

Similar to the differences found in theIEA between both groups, a higher c-IMTwas seen in type 1 diabetic patientscompared with nondiabetic subjects(0.70 6 0.21 vs. 0.58 6 0.20 mm; P =0.05), and this parameter correlated sig-nificantly with VCAM-1 protein levels inthe epigastric artery (r = 0.220, P = 0.046).Finally, the c-IMT also showed a signifi-cant correlation with fasting glucose lev-els (r = 0.283, P = 0.009) and the degree of

arterial lumen reduction (r = 0.307, P =0.02).

CONCLUSIONSdThis cross-sectionalstudy demonstrates, for the first time, thattype 1 diabetes produces a proinflam-matory state in the artery wall of uremicpatients, evidenced by the expression ofcytokines and adhesion molecules aswell as a greater degree of NFkB-p65 ac-tivation, which are related to more severevascular lesions and higher c-IMT. Tobetter elucidate the role of type 1 diabetesin atherosclerosis, we excluded patientswith type 2 diabetes and used as a controlgroup of uremic patients without diabe-tes. We focused on candidate patients fortransplant to avoid including patientswith a higher cardiovascular morbidityand who were therefore not eligible fortransplantation. Thus, our results highlightthe role of inflammatory pathways in thedevelopment of atheromatosis in type 1diabetic patients with uremia.

Atheromatosis is the result of a com-plex inflammatory process in the arterywall where important risk factors, such asdiabetes and uremia, may play a crucialrole via different pathogenetic mecha-nisms. The relative contribution of eachfactor to the progression of atheromatosiswhen both pathological situations concur

Figure 1dHistological and immunohistochemical characteristics of the IEA. The intima thickening is composed of SMA-positive cells (magnifi-cation 3200) (A) and collagen fibers positive for Sirius Red (magnification 3100) (B). Macrophages only are detectable as isolated cells in theintima (magnification3400) (C) or forming aggregates around medial calcifications (magnification3100) (D). Abundant localization of MCP-1–positive cells (E), as well as IL-6–positive cells (F), is present in both the intima and muscular layers. Strong VCAM-1 expression is observed in theendothelial layer (G) of the artery wall (magnification3400). Note that endothelial and SMCs, as well as SMA-positive cells in the neointima, arepositive for the three molecules. A and B represent contiguous sections of the same artery; note the fibrous cap formed by SMA-positive cells andcollagen. Arrow, internal elastic lamina; L, lumen. (A high-quality digital representation of this figure is available in the online issue.)

Figure 2dA: Differences in the gene expression of proinflammatory markers in the artery wall oftype 1 diabetic patients (-) and nondiabetic patients (▫). B: Differences in the quantification ofproinflammatory proteins between type 1 diabetic patients (-) and nondiabetic patients (▫).*P , 0.05; **P , 0.01.

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is, however, undetermined. CKD is asso-ciated with increased generation of cyto-kines and adhesion molecules, suggestingan early endothelial dysfunction and up-regulation of these molecules in the arterywall (18,19). Growing evidence indicatesthat proinflammatory cytokines play a de-terminant role in the development of vas-cular diabetes complications, especially inthe presence of high glucose levels (9).Thus, it is plausible that the negative ef-fects of both risk factors on the artery wallcan be enhanced. In other words, diabetesmay accelerate the development of ath-erosclerosis in the presence of uremia bytriggering inflammation pathways.

We focused our attention on thestudy of the gene expression and proteintranscription of proinflammatory cyto-kines (IL-6 and MCP-1) and adhesionmolecules (ICAM-1 and VCAM-1), factorspotentially involved in the pathogenesis ofatheromatosis. Indeed, a higher expres-sion of IL-6, MCP-1, and adhesion mole-cules was observed in the artery wall oftype 1 diabetic patients compared withnondiabetic subjects. Activation of theseinflammatory molecules could translate tomore severe vascular lesions (fibrosis andluminal narrowing) and a higher c-IMT, aswas observed in our diabetic patients. Infact, overexpression of IL-6 andMCP-1wasrelated with more advanced atheromatouslesions, endorsing the role of MCP-1 both

for attracting monocytes to the inflamma-tion area and inducing collagen produc-tion by SMA-positive cells (20). Inaddition, c-IMT measurements correlatedwith VCAM-1 protein levels and the de-gree of arterial lumen reduction, whichsuggests that this parameter may be anearly surrogate marker of atheromatosisin diabetic patients who are candidatesfor kidney transplantation.

NFkB is a ubiquitous transcriptionfactor that regulates the inflammatory re-sponse and whose overexpression seems tobe related to type 1 diabetes (18,19).Thus, a greater NFkB expression in thearterial wall of diabetic patients seemslikely. Accordingly, a significantly highernuclear localization of p65 was seen in ourtype 1 diabetic patients, supporting therole of this important transcription factorin the atheromatous process through theincrease of proinflammatory cytokines. Inconsonance with this finding, we alsoobserved a significant correlation be-tween p65 nuclear translocation and lev-els of MCP-1 in the type 1 diabetic groupbut not in nondiabetic subjects. This sug-gests that, although MCP-1 may be regu-lated in an NFkB-dependent fashion inthe presence of a proinflammatory statelike type 1 diabetes (21), other cytokinesmay be under the control of more com-plex interactions, especially when diabe-tes is absent.

Of note, histological analysis of ourpatients showed that intimal thickeningof the IEA was mostly composed of SMA-positive cells and collagen fibers but notof CD68-positive macrophages. Thus, apathogenicmechanism accounting for therole of proinflammatory cytokines pro-duced by resident and infiltrating cells,in patients with diabetes and uremia,seems plausible. In early stages of athero-matosis, intima cells recruit SMCs andSMC precursors by the release of multiplefactors (cytokines and inflammatorymarkers). Later, these cells may switchfrom a contractile phenotype to a synthe-tic phenotype, generating in situ cytokines(IL-6 andMCP-1) and adhesionmolecules(VCAM-1). Finally, these molecules in-duce the generation of fibrosis and arteriallumen reduction, contributing to perpet-uating the inflammatory environment inthe lesion, as previously documented (22).The fact that the production of MCP-1,IL-6, and VCAM-1 correlated with moresevere vascular lesions in the presence ofabundant SMA-positive cells and collagenfibers in the intima layer of our patientssupports this view.

Previous histological reports fromautopsies of elderly individuals who diedof noncardiac diseases have documentedthat coronary arteries and the IEA undergoprogressive narrowing with age as a resultof intimal thickening, with migration of

Figure 3dImmunofluorescence images showing the difference in the expression and nuclear localization of NFkB-p65 in the artery wall of the IEAfrom a nondiabetic patient (A) and a type 1 diabetic patient (B) with a similar grade of intima thickening and CKD. The nuclei were stained withDAPI (blue), and NFkB-p65 was revealed using an Alexa-conjugated antibody (red). The third column shows the colocalization. Original mag-nification3600. The histogram represents the increase in percentage of colocalization referred to the control (**P, 0.001). DM, diabetes. (A high-quality digital representation of this figure is available in the online issue.)

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myocytes from the media and duplicationof the internal elastic lamina. These fea-tures were present in all the musculararteries studied, and the internal thoracicartery, a mainly elastic vessel, was the onlyartery resistant to atherosclerosis (23,24).Although this evidence favors the inter-pretation that our findings in the IEA arerelevant to the understanding of the vas-cular lesions present in other territorieswith predominantly muscular walls, wecannot be sure that they are generalizableto all arterial territories. A certain degree ofcontroversy remains, because older stud-ies had proposed the IEA as a good sourcefor coronary artery bypass grafts based onits relative resistance to atheromatosis(25). Although no intimal calcification ofthe IEA was found in our study, most dia-betic patients had a significant arteriallumen reduction (Supplementary Table 2).By contrast, a higher proportion of thenondiabetic patients had minimal luminalnarrowing (,5%). Our molecular and cellbiology studies of the IEA suggest that lo-cal vascular injury induced by the combi-nation of diabetes anduremiamaypromotethe overexpression of proinflammatorymolecules, leading to more advanced ath-eromatous lesions in the IEA, which agreeswith the carotid artery thickening detectedby ultrasounds.

A higher prevalence and severity ofmedial calcification also was observed intype 1 diabetic patients. In agreement withprevious reports (26), we found no rela-tionship between the molecules studiedand these lesions. Of interest, fasting glu-cose levels were associated with medial-layer calcification in these patients. Thus,advanced glycation end products and he-modynamic abnormalities related to type1 diabetes may trigger signaling pathwaysinvolved in the appearance of vascular cal-cification (27). Glycemic control was ac-ceptable (HbA1c ,7%) in 60% of ourdiabetic patients before starting dialysis,but we cannot rule out that a longer dura-tion of diabetes could contribute to moresevere vascular lesions. The fact that thediabetic patients were older in our studysupports this argument.

Whether modulation of the inflam-matory response by anti-inflammatorydrugs may slow down the atheromatousprocess in type 1 diabetic patients, as ob-served in other diabetes complications(28), needs be tested in future studies.

This study has some limitations. First,we did not determine the plasma levels ofthe cytokines studied. Plasma cytokinelevelsmay vary greatly under certain clinical

conditions, such as diabetes and uremia.Thus, direct assessment of proinflamma-tory gene activation in vascular tissue maybetter reflect the pathways of inflamma-tory-related atheromatosis, especially indiabetic patients. In addition, we con-ducted a rigorous and accurate molecularand histological analysis, which affordsreliability to our findings. Second, we didnot assess proinflammatory cytokine ex-pression in the artery wall of healthysubjects, but our objective was to evaluatethe effect of type 1 diabetes on the athe-romatosis-related inflammatory state inkidney transplantation candidates, anduremia was present in all the patients. Inaddition, we cannot rule out that the othermolecules undetermined in this study,such as adipokines, angiotensin II, orcoagulation factors, may have a relevantrole in the pathogenesis of atheromatosis.Nevertheless, this was not the scope of ourstudy, and it deserves additional researchto elucidate alternative mechanisms of di-sease. Finally, because the sample sizemaybe considered small, the results of thisstudy should be taken with caution, andadditional studies are required to enhancethe understanding of atheromatosis-relatedinflammation in the presence of diabetesand uremia.

In conclusion, the findings of thisstudy show that proinflammatory cyto-kines, adhesion molecules, and NFkB mayplay a pivotal role in the atheromatousprocess in type 1 diabetic patients withCKD. These results provide potentialmechanistic insights for the increased in-flammation and accelerated atherosclerosisin this particular population. The under-standing of these mechanisms may lead tonew therapeutic strategies in this high-riskpopulation.

AcknowledgmentsdThis study was sup-ported by the Spanish Ministry of Educationand Science (grant no. SAF2007-60314) andin part by the Instituto de Salud Carlos IIIRETIC (grant nos. RD06/0016 [RedInRen],FIS PI07/0732, and FIS PI10/01020 from theSpanish Ministry of Science and Innovation[MICINN]), and the Consejería de Salud delGobierno de Andalucía (grant no. PI-0499/2009), Fondos Europeos para el DesarrolloRegional.No potential conflicts of interest relevant to

this article were reported.J.T. and E.S. contributed to writing the

manuscript, obtained the research data for geneand protein expression, and contributed to thehistological and inmmunohistochemical analy-sis. J.F. conducted the carotid echography. M.R.

and J.M.G.-P. obtained all the clinical data. A.T.contributed to the critical review of the man-uscript. D.H. is the guarantor for this article,participated in the study design, performed thestatistical analysis, and wrote the manuscript.The authors thank Ian Johnstone, from the

University Hospital Carlos Haya, for linguisticassistance. The authors also thank the Can-arian renal transplantation team for their col-laboration.

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