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Original article 747

Accelerated arterial stiffening
and gene expression profile ofthe aorta in patients with coronary artery diseaseCeline Fassota, Marie Brietb, Philippe Rostagnoc, Pascal Barbryc,Claudine Perreta, Dominique Laudea, Pierre Boutouyriea, Erwan Bozeca,Patrick Brunevale, Christian Latremouilled and Stephane Laurenta
Background Hypertension and chronic renal failure

(CRF) are considered models of accelerated arterial

stiffening. Arterial stiffness increases further when CRF is

associated with hypertension. We hypothesized that, in

patients with mild CRF, aortic gene expression profile would

include genes involved in arterial calcifications and

enlargement.

Method We analysed human aorta with the ‘GeneChip

Microarray’ technology, in patients with or without CRF,

scheduled for a coronary artery bypass graft.

Results Nine of 25 patients had high-quality RNA and were

included in the study. Among the 101 transcripts

differentially expressed between CRF patients and controls,

97 transcripts were overexpressed in CRF patients. Two

genes had the highest overexpression in CRF patients:

lumican (LUM), involved in the regulation of collagen

fibrillogenesis; and ornithine decarboxylase (ODC1),

involved in polyamine biosynthesis, smooth muscle cell

growth and proliferation. Immunohistochemical staining

revealed an increased amount of LUM and ODC1 in the

vascular smooth muscle cells (VSMCs) of CRF compared to

non-CRF aortic sections. Eight genes were implicated in the

regulation of the cytoskeleton (including capping protein

muscle Z-line 1 a and moesin) and cell migration, and five

genes were implicated in extracellular matrix function and

apoptosis. A trend towards an upregulation of candidate

genes involved in arterial calcifications was observed in

CRF patients, but did not reach statistical significance.

Carotid-femoral pulse wave velocity was not correlated with

gene expression level.

opyright © Lippincott Williams & Wilkins. Unauth

0263-6352 � 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins

Conclusion In conclusion, these results show that patients

at an early stage of CRF have a specific gene expression

profile of aortic tissue and suggest that genes implicated in

collagen fibrillogenesis, and VSMCs migration and

proliferation, particularly LUM and ODC1, may play a role.

J Hypertens 26:747–757 Q 2008 Wolters Kluwer Health |

Lippincott Williams & Wilkins.

Journal of Hypertension 2008, 26:747–757

Keywords: aorta, arterial remodelling, arterial stiffness, kidney disease,microarray

Abbreviations: ALPL, alkaline phosphatase; APOD, apolipoprotein D;BGLAP, osteocalcin CAD, coronary artery disease; CAP1, adenylatecyclase-associated protein 1; CAPZA1, capping protein muscle Z-line 1alpha; CCA, common carotid artery; COL I, collagen I; COL II, collagen II;CRF, chronic renal failure; CV, cardiovascular; ESRD, end-stage renaldisease; ET1, endothelin 1; GFR, glomerular filtration rate; HCT,hypercholesterolemia; LEP, leptin; MDRD, Modification of Diet in RenalDisease; MGP, matrix gla protein; MI, myocardial infarction; MSN, moesin;ODC1, ornithine decarboxylase; OPG, osteoprotegerin; PWV, pulse wavevelocity; SERPIN 2, serine proteinase inhibitor; SMC, smooth muscle cell;SSP1, osteopontin; VSMC, vascular smooth muscle cell

aFaculty of Medicine, University Paris-Descartes, Department of Pharmacology,Georges Pompidou European Hospital, and Institut National de la Sante et de laRecherche Medicale U652, bDepartment of Physiology, Georges PompidouEuropean Hospital, Paris, cLaboratory of Physiological Genomics, Sofia-Antipolis,dDepartment of Cardiac Surgery, Georges Pompidou European Hospital andeDepartment of Pathology, Georges Pompidou European Hospital, and InstitutNational de la Sante et de la Recherche Medicale U652, Paris, France

Correspondence to Stephane Laurent, MD, PhD, Department of Pharmacologyand Institut National de la Sante et de la Recherche Medicale U652, HopitalEuropeen Georges Pompidou, 20 rue Leblanc, 75015 Paris, FranceTel: +33 1 56 09 39 91; fax: +33 1 56 09 39 92;e-mail: [email protected]

Received 25 August 2007 Revised 11 November 2007Accepted 23 November 2007

IntroductionArterial stiffness and wave reflections are now well

accepted as the most important determinants of increasing

systolic and pulse pressures in ageing societies [1]. Hyper-

tension and chronic renal failure (CRF) are considered

models of accelerated arterial stiffening [1–3]. We recently

showed that, compared to uncomplicated hypertension,

arterial stiffness further increased when mild-to-moderate

CRF was associated with hypertension [4]. The molecular

mechanisms of arterial stiffening have been well described

in hypertension [2,5] and end- stage renal disease (ESRD)

[3,6], but little in mild-to-moderate CRF.

CRF patients have an increased risk of cardiovascular

morbidity and mortality [7], correlated to the decrease of

glomerular filtration rate (GFR) [8] that impairs cardiac

and arterial functions. Pathogenic mechanisms of arterial

disease in CRF include endothelial dysfunction [9] lead-

ing to vasoconstriction and arterial growth, oxidant stress

[10], dysfunction of nitric oxide metabolism [11], increase

in inflammatory factors [12,13], and vascular calcifications

[6,14]. Vascular calcifications in ESRD patients have

strong predictive value for cardiovascular and all-cause

mortality [15]. A likely mechanism is the association with

an increased stiffness of large, capacitive, elastic-type

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748 Journal of Hypertension 2008, Vol 26 No 4

arteries such as the aorta and the common carotid artery

[15]. Vascular calcifications occur more often and at an

earlier age in patients with ESRD than in normal controls.

We previously showed that arterial enlargement and

increased arterial stiffness occur in parallel with the

decline in renal function in patients with mild-to-mod-

erate CRF [4].

We hypothesized that, in patients with mildly altered

renal function, the gene expression profile of the aorta

would include genes involved in arterial calcifications and

remodelling. We used punch biopsies of patients sched-

uled for coronary artery bypass surgery and a microarray

technology [16] to characterize the gene expression pro-

file of aortic tissue. We compared patients with mild-to-

moderate CRF to patients with normal renal function,

and studied the relationship between aortic stiffness,

measured through carotid-femoral pulse wave velocity

(PWV), and underexpressed or overexpressed genes.

Materials and methodsPatientsTwenty-five patients, with and without CRF, scheduled

for coronary artery bypass surgery, were recruited at the

Department of Cardiac Surgery of Hopital Europeen

Georges Pompidou. The study was conducted according

to the guidelines of the ethics committee of the institu-

tion. We included patients with coronary artery disease

(CAD) because they were carrying an ischemic cardio-

myopathy without an associated disease that was able to

compromise the interpretation of the results. Demo-

graphic data with details of cardiovascular risk factors

and blood samples were collected on the day when aortic

stiffness was measured, that is the day before the bypass

surgery. Hypertension, diabetes and hypercholesterole-

mia (HCT) were indicated by a previous diagnosis or the

use of antihypertensive, oral hypoglycaemic or choles-

terol-lowering agents, respectively. Mild renal insuffi-

ciency was due to hypertension and/or diabetes, and

not other disease.

Glomerular filtration rate measurementsThe Modification of Diet in Renal Disease (MDRD)

simplified equation was used to estimate GFR [17]:

Glomerular filtration rateðGFRÞ½ml=min per 1:73m2�

¼ 186� ½serum Crðmg=dlÞ��1:154 � ½age��0:203

� ½0:742 if female� � ½1:210 if black�

Pulse wave velocity measurementsAortic stiffness was estimated by carotido-femoral PWV,

measured along the descending thoraco-abdominal

aorta by the foot-to-foot velocity method, as previously

published and validated [18]. Briefly, waveforms were

obtained transcutaneously over the right common

opyright © Lippincott Williams & Wilkins. Unautho

carotid artery (CCA) and femoral artery, and the time

delay (t) was measured between the feet of the two

waveforms.

We adjusted PWV on age, HCT and previous myocardial

infarction (MI) using a general linear model to determine

the multivariate b coefficients in a stepwise manner and

to calculate the predicted values on the basis of the

model. In a multivariate analysis of the 25 patients,

age, HCT and MI were independently correlated to

PWV (P< 0.05 each), and explained, in stepwise

regression analysis, 28, 16 and 31% of the variance (R2

increment) of PWV, respectively.

Aortic biopsiesThe punch biopsies (one to three samples per patient)

taken at the time of the coronary bypass surgery

were immediately conserved in RNAlater (Qiagen, Cour-

taboeuf, France) for RNA analysis or frozen in liquid

nitrogen for western blot and stored at �808C until

processing, or fixed in formaldehyde for immunohisto-

chemistry. In four patients, a pilot histological study has

shown a normal ascending aorta, without atherosclerosis

and overt calcifications. The thickness of the intima was

normal. The media was normal, with a regular organiz-

ation of elastin lamellae and smooth muscle cells (SMCs).

The adventitia was normal. These findings strongly

suggest that the RNA, extracted from the punch biopsy,

is representative of a normal SMC population, rather than

an atherosclerotic cell population, even in CRF patients.

Aortic tissue was taken in a young organ donor during

transplantation, to serve as reference. Indeed, the extent

of atherosclerosis was limited in this subject.

RNA extraction and amplificationAfter extraction of total RNA from human aortic samples

by TRIzol reagent (Invitrogen, Cergy Pontoise, France),

quality and concentration were checked by the Agilent

Technology. RNA with a good quality was amplified

by the Amino Allyl MessageAmp aRNA kit (Ambion,

Huntingdon, UK), according to the manufacturer’s

instructions.

Microarray analysisThe oligonucleotide microarrays were supplied by the

laboratory of Physiological Genomics of Sophia-Antipo-

lis. These arrays contained 27 648 oligonucleotides

probes (length � 51 bp) specific to 25 279 human tran-

scripts. The list of the probes is available online (http://

www.microarray.fr:8080/mediante/index). Antisens RNA

was coupled with Cy3 and Cy5 postlabelling reactive

dyes (Amersham, Little Chalfont, UK). After a postpro-

cessing step, microarrays were hybridized with labelled-

RNA as previously described [19]. Slides were scanned

with a ScanArray 5000 (GSI Lumonics, Munich,

Germany) and images were quantified with the GenePix

Pro 5.0 program (Axon Instruments, Chatillon, France).

rized reproduction of this article is prohibited.

http://www.microarray.fr:8080/mediante/index

http://www.microarray.fr:8080/mediante/index

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Aortic gene expression profile Fassot et al. 749

Fig. 1

27648 oligonucleotides probes

17 897 oligonucleotides probes

1716 probes

101 probesDifferentially expressed between CRF+ and CRF--

Gene expresion level > 500

Present in all samples (n = 9)

Wilcoxon testP < 0.05

97 probesOverexpressed in CRF+

≥ 1.5-fold

Scheme for analysis of profiling data. CRF, chronic renal failure.

On each slide of microarray, we hybridized the aRNA of

a patient and the reference (donor). This technique

allowed us to normalize values of patients to reference

values and then compare patients between them. More-

over, because incorporations of Cy3 and Cy5 dyes can

differ significantly, data were further normalized using a

dye-swap method [20]. Statistical analysis was performed

using Microsoft Excel 2003 functions as described in

Fig. 1.

Protein analysisImmunohistochemistry

The proteins encoded by differentially expressed genes

were studied by immunohistochemistry on aortic biop-

sies of CRF and non-CRF patients. Paraffin-embedded

sections were treated with xylen, fixed for 10 min in cold

acetone and kept wet in Tris-buffered saline (TBS)

until processing. All antigens were detected with very

well characterized monoclonal [antidynein (Santa-Cruz

Biotechnology, Santa-Cruz, USA), moesin (MSN; Lab

Vision Corporation, Fremont, USA)] and polyclonal

[anti-CAPZA1 (Chemicon, Chandlers Ford, UK),

ornithine decarboxylase (ODC1; Santa-Cruz Biotechnol-

ogy, Santa-Cruz, USA), osteoprotegerin (OPG), osteocal-

cin and lumican (LUM; R&D Systems, Lille, France)]

antibodies. Dilutions used were done according to the

manufacturer’s instructions. Antigen presence was

revealed by the indirect immunoperoxidase method.

Western blot

A western blot analysis of proteins of interest was per-

formed following standard techniques. Proteins were

extracted from arterial biopsies of CRF and non-CRF


patients. Total protein content was determined by

the Bradford technique [21]. Equal amounts (50 mg) of

the denatured proteins were loaded per lane, separated

on a Ready Gel 10% Tris–HCl polyacrylamide gel

(Bio-Rad, France) and transferred to a nitrocellulose

membrane. Membranes were incubated with mono-

clonal antibodies directed against MSN or polyclonal

antibodies against OPG. Subsequent analysis used an

antimouse, an antigoat or an antichicken IgG peroxidase

complex, as a second antibody (dilutions 1 : 3000–

1 : 5000), and chemiluminescence emitted from luminol

oxidized by peroxidase as a detection method (ECL

System, Amersham).

ResultsPatientsOn the basis of their GFR, the 25 patients were split into

equal tertiles: lowest GFR (37–68 ml/min per 1.73 m2),

medium GFR (69–91 ml/min per 1.73 m2) and highest

values (greater than 92 ml/min per 1.73 m2) (Table 1).

The patients of the lowest tertile were considered as

CRF population and the others as non-CRF population.

These two groups were not significantly different, as far

as sex ratio, brachial SBP, DBP, MBP, PP and PWV were

concerned (Table 1). In the CRF population, time from

the beginning of the disease was 10� 4 years.

According to the quantity and quality of the extracted

RNA, nine patients, including four non-CRF patients

and five CRF patients, were analysed on oligonucleotides

microarray. The clinical characteristics of the total popu-

lation, the nine patients used for microarray analysis and

the five patients used for protein analysis, are presented

in Table 1.

Analysis of genes involved in arterial calcificationWe first tested the hypothesis that genes encoding

proteins implicated in vascular calcifications would be

differentially expressed between aortic tissues of patients

with CRF compared to patients without CRF. Although

the expression level of genes encoding proteins involved

in vascular calcifications [matrix Gla protein: MGP; osteo-

pontin: SSP1; osteoprotegerin: TNFRSF11B; osteocal-

cin: BGLAP; and osteo/chondrocytic transcription factors

(Sox9, Runx2 and Msx2); alkaline phosphatase: ALPL;

leptin: LEP; collagen I: COL I; collagen II: COL II]

was 1.2–2.2-fold higher than in non-CRF patients, no

significant difference was observed between groups

(Table 2).

Differential gene expression of the complete data setThe complete data set was filtered to remove transcripts

with low expression level (less than 500) and not present

in all of the nine samples, yielding 1716 genes (Fig. 1).

Statistically significant changes in gene expression were

identified using a Wilcoxon test with a P-value less than

0.05. There were 101 genes differentially expressed


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Table 1 Clinical data of patients, in the whole population according to tertiles of glomerular filtration rate (GFR), and in subpopulations forgene and protein analysis

Parameters

Total population(n¼25)

Subpopulation for geneanalysis

Subpopulation for proteinanalysis

First tertile Second tertile Third tertile CRF� CRFþ CRF� CRFþ

N 8 9 8 4 5 3 2Age (years) 66�4§ 66�2§§ 56�2 57�2 68�10 59�12 58�13Sex ratio (M/F) 8/0 8/1 8/0 4/0 5/0 3/0 2/0Height (cm) 173�1 172�2 173�3 173�7 173�2 168�5 176�8Weight (kg) 76�4 79�4 73�10 86�19 73�9 74�11 97�0BMI (kg/m2) 26.5�1.5 26.4�0.8 26.8�1.3 28.5�4.3 24.7�3.3 25.9�2.2 31.4�3.0GFR, MDRD (ml/min/1.73 m3) 56.0�3.7§§§ 83.5�2.4§§§ 104.4�4.8 98.7�23.5 57.5�7.5MM 92.9�5.9 45.9�13.1MM

From the beginning of CRF (years) 10 �4 / / / / /Hypertension (yes/no) 6/2 7/2 5/3 2/2 3/2 2/1 2/0HCT (yes/no) 5/3 8/1 7/1 3/1 3/2 2/1 1/1Previous MI (yes/no) 3/5 6/3 3/5 2/2 1/4 1/2 1/1Diabetes (yes/no) 4/4 4/5 4/14 2/2 2/3 0/3 1/1Smoking (current/past or never) 1/7 1/8 2/6 1/3 0/5 1/2 1/1SBP (mmHg) 127�4 132�6 120�7 130�16 132�11 130�3 120�11DBP (mmHg) 70�4 71�2 68�3 70�9 72�13 69�6 66�11MBP (mmHg) 90�3 90�3 85�4 90�11 92�11 87�6 86�9PP (mmHg) 57�4 61�5 52�5 61�10 60�12 61�6 55�1HR (bpm) 70�5 63�4 64�3 63�9 75�17 71�3 67�9Adjusted PWV (m/s)a 14.7�0.8 17.8�1.3 16.3�1.0 17.6�3.1 14.0�2.5 16.2�4.7 15.3�1.1

Mean�SD of total population divided in three groups according to GFR tertiles (§P<0.05 vs. third tertile, §§P<0.01 vs. third tertile, §§§P<0.001 vs. third tertile) andsubpopulation for gene analysis and protein analysis (MMP<0.01 vs. CRF�). CRF, chronic renal failure; DBP, diastolic blood pressure; HCT, hypercholesterolemia; HR,heart rate; MBP, mean blood pressure; MDRD, Modification of Diet in Renal Disease; MI, myocardial infarction; PP, pulse pressure; PWV, pulse wave velocity; SBP, systolicblood pressure. a Adjusted PWV: adjustment to age, HCT and previous MI.

between CRF and non-CRF samples and 97 of them with

an absolute fold change of 1.5 at least (Fig. 1). The full list

of 101 genes is presented in Table 3.

Two genes had the highest overexpression in CRF

patients: LUM and ODC1. LUM, upregulated 3.0-fold,

is principally involved in the regulation of collagen

fibrillogenesis [22]. ODC1 (overexpressed 3.1-fold),

involved in polyamine biosynthesis, and apolipoprotein

D (APOD, upregulated two-fold) play an essential role in

SMC growth and proliferation (Table 3, Fig. 2). We

identified eight genes implicated in the regulation of

the cytoskeleton. Four genes are known to interact with


Table 2 Evolution of gene expression profile of the aorta, concerningcalcifications, in patients with (CRFR) or without (CRFS) chronic renal

Gene Description

Inhibitors of vascular calcificationMGP Matrix Gla proteinSPP1 OsteopontinTNFRSF11B OsteoprotegerinSPARC OsteonectinBGLAP Osteocalcin

Activators of vascular calcificationRUNX2 Osteo/chondrocytic transcription factorMSX2 Osteo/chondrocytic transcription factorSOX9 Osteo/chondrocytic transcription factorBMP2 Bone morphogenic protein 2ALPL Alkaline phosphataseLEP LeptinCOL I Collagen ICOL II Collagen II

Implicated in vascular calcificationLUM Lumican

MP<0.05.

actin [capping protein muscle Z-line 1 a (CAPZA1);

MSN; actin-related protein 2/3 complex (ARPC3); and

adenylate cyclase-associated protein 1 (CAP1)] and four

genes are known to interact with tubulin [tubulin-specific

chaperone A (TBCA); heat shock protein 105 (HSPH1); a

protein similar to myomegalin (LOC200019); and dynein

(DNCH1)] (Fig. 2). We also detected two other genes

implicated in matrix function [serine proteinase inhibitor

(SERPIN 2) and a protein similar to fibronectin type 3

(MGC 45564)] as well as three genes involved in apop-

tosis [calgranulin A (S100A8) DEAD box polypeptide 47

(DDX47) and small subunit of the calpain (CAPNS2)]

(Fig. 2).


genes encoding for proteins regulating or implicated in vascularfailure

Fold change (CRFþ/CRF�) P

1.6 NS1.4 NS1.2 NS1.0 NS1.5 NS

1.6 NS1.7 NS1.2 NS1.1 NS1.6 NS1.5 NS1.3 NS2.2 NS

3.0 M

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.


Table 3 One hundred and one transcripts differentially expressed between aorta of chronic renal failure (CRF) and non-CRF patients

Gene Description Fold change Category

STK3 Serine/threonine kinase 3 1.6 Signalling/communicationMRGX4 MAS-related gene MRGX4 1.6 Signalling/communicationIFNA2 Interferon a 2 1.5 Cell defenseTRIP15 Thyroid receptor interacting protein 15 1.8 Signalling/communicationPCDHB16 Protocadherin b16 1.6 Signalling/communicationAIM1 Absent in melanoma 1 1.6 Signalling/communicationBACH Brain acyl-CoA hydrolase 1.6 MetabolismLOC378344 Similar to riken cDNA 6332401019 gene 1.7 UnclassifiedLOC200019 Similar to myomegalin 1.5 Cell structure/motilityMGC45564 Similar to fibronectin type 3 1.6 Cell structure/motilityMGC31963 Kidney predominant protein NCU-G1 1.8 UnclassifiedFLJ21128 Hypothetical protein FLJ21128 1.7 UnclassifiedNUDT11 Nucleoside diphosphate linked moiety X m 11 2.2 Gene/protein expressionPRDM10 PR domain containing 10 1.9 Gene/protein expressionMANBA Mannosidase ba lysosomal 1.8 MetabolismBCL9 B-cell CLL/lymphoma 9 1.8 Signalling/communicationPIGA Phosphatidylinositol glycan, class A 1.9 Signalling/communicationRFWD1 TNF receptor associated factor 7 1.5 Cell divisionDNCH1 Dynein cytoplasmic heavy polypeptide 1 1.8 Cell structure/motilityC20orf11 Chromosome 20 open reading frame 11 2.1 Gene/protein expressionFLJ32752 Hypothetical protein FLJ32752 2.0 UnclassifiedGGA1 Golgi ass. g adaptin ear cont. ARF bind. prot. 1 1.5 Gene/protein expressionSENP7 Sentrin-specific protease 7 1.6 Gene/protein expressionARPC3 Actin-related protein 2/3 complex 1.7 Cell structure/motilityTPK1 Thiamin pyrophosphokinase 1 1.6 Cell structure/motilityLOC158471 Hypothetical protein BC019095 1.8 MetabolismFLJ36664 Hypothetical protein FLJ36664 1.9 UnclassifiedLOC150035 cDNA FLJ11616 fis 1.9 UnclassifiedZNF304 Zinc finger protein 304 1.6 Gene/protein expressionLALP1 Lysosomal apyrase-like protein 1 1.6 Cell divisionKIAA1107 Unknow protein mRNA, partial cds 1.5 UnclassifiedMSN Moesin 1.8 Cell structure/motilityZNF225 Zinc finger protein 225 1.7 Gene/protein expressionFLJ14816 Hypothetical protein FLJ14816 1.6 UnclassifiedH1F0 Histone family 1.8 Cell divisionTRIP3 Thyroid hormone receptor interactor 3 2.1 Signalling/communicationIRF1 Interferon regulatory factor 1 1.9 Gene/protein expressionTLOC1 Translocation protein 1 1.7 Signalling/communicationMGC16169 Hypothetical protein MGC16169 2.1 UnclassifiedMGC26610 Hypothetical protein MGC26610 1.7 UnclassifiedKSP37 Ksp 37 protein 1.5 Cell defenseSERPIN2 Serine proteinase inhibitor, clade E, member 2 1.7 Signalling/communicationKIAA1677 cDNA DKFZp434M0516 1.8 UnclassifiedCLECSF1 C-type lectin, superfamily member 1 (cart-der) 1.9 UnclassifiedDKFZp451J0118 DKFZp451J0118 protein 1.7 MetabolismFLJ21816 Hypothetical protein FLJ21216 1.9 Signalling/communicationPME-1 Protein phosphatase methytransferase 1 1.7 Gene/Protein expressionINSRR Insulin receptor-related protein 1.6 Cell structure/motilityC20orf155 Chromosome 20 open reading frame 155 1.6 Gene/protein expressionCAP1 Adenylate cyclase associated protein 1 1.5 Cell structure/motilityEBAF Endometrial bleeding associated factor 1.3 Signalling/communicationZNF18 Zinc finger protein 18 1.4 Gene/protein expressionDDX47 DEAD box polypeptide 47 2.1 Cell divisionFUT4 Fucosyltransferase 4 1.9 MetabolismPAIP2 Polybinding protein interacting protein 2 2.1 Signalling/communicationCOX7C Cytochrome c oxidase subunit viic 2.3 MetabolismCAPZA1 Capping protein muscle Z-line a 1 2.1 Cell structure/motilityAPOD Apolipoprotein D 2.0 MetabolismTUWD12 Acetylgalactosaminyltransferase 2.0 MetabolismKR19 Zinc finger protein 19 1.8 Gene/protein expressionHMGN2 High-mobility group nucleosomal-binding domain 2 2.6 Cell divisionUBN1 Ubinuclein 1 1.7 Signalling/communicationTBCA Tubulin-specific chaperone a 2.1 Cell structure/motilityTEAD2 TEA domain family member 2 1.5 Gene/protein expressionSTK38L Serine/threonine kinase 38-like 1.5 Gene/protein expressionTM4SF1 Transmembrane 4 superfamily member 1 1.7 Signalling/communicationFLJ25157 Hypothetical protein FLJ25157 1.8 Signalling/communicationTAF10 Taf 10 RNA polymerase II 1.9 UnclassifiedC15orf15 Chromosome 15 open reading frame 15 2.2 Gene/protein expressionSTC2 Stanniocalcin 2 1.9 Gene/protein expressionALS2CR17 Amyotrophic lateral sclerosis 2 chromosome region 17 2.5 Cell defenseODC1 Ornithine decarboxylase 1 3.1 MetabolismLUM Lumican 3.0 Cell structure/motilityHM13 Histocompatibility 13 2.4 Cell structure/motilityPOLS Polymerase sigma 2.1 Cell defense

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Table 3 (continued )

Gene Description Fold change Category

C14orf92 Chromosome 14 open reading frame 92 2.0 Gene/protein expressionZNF211 Zinc finger protein 211 1.7 Gene/protein expressionZNF217 Zinc finger protein 217 1.9 Gene/protein expressionSSX2 Synovial sarcoma X2 1.9 Gene/protein expressionSFRS7 Splicing factor 1.7 Gene/protein expressionFLJ10634 Hypothetical protein FLJ10634 1.9 UnclassifiedPIP3AP Myotubularin-related protein 12 2.0 Signalling/communicationCAPNS2 Calpain small subunit 2 2.1 Gene/protein expressionLOC340371 Hypothetical protein LOC340371 2.2 UnclassifiedRPS6KC1 Ribosomal protein S6 kinase 1.6 Gene/protein expressionMAPK10 MAP kinase 10 1.9 Signalling/communicationC18orf1 Chromosome 18 open reading frame 1 1.7 Gene/protein expressionARL7 ADP-ribosylation factor-like 7 1.8 Gene/protein expressionCCL11 Eotaxin 1 1.6 Signalling/communicationMGC20446 Hypothetical protein MGC20447 1.7 UnclassifiedNYX Nyctalopin 1.8 Signalling/communicationS100A8 Calgranulin A 2.1 Cell divisionRALBP1 Rala-binding protein 1 1.4 Signalling/communicationHSPH1 Heat shock protein 105 1.4 Cell structure/motilityTBX19 T-box 19 1.9 Gene/Protein expressionTJP4 Tight junction protein 4 1.7 Signalling/communicationCSNK2A2 Casein kinase 2 2.0 Signalling/communicationNEURL Neuralized homolog like 1.8 Cell divisionIL13RA1 Interleukin 13 receptor a1 2.0 Signalling/communicationUBE2J2 Ubiquitin-conjugated enzyme E2, J2 1.7 Gene/protein expressionHCLS1 Hematopoietic cell-specific Lyn substrate 1 1.8 Cell defense

Functional classification of differentially expressedgenesWe classified the 101 transcripts differentially expressed

between groups, according to the nine functional

categories described by Hwang et al. [23] (Fig. 3). As a

group, transcripts encoding proteins potentially involved

in the mechanical regulation of vascular function (‘sig-


Fig. 2

Level of expression of genes selected for their implication in matrix, migrati

nalling/communication’ and ‘structure/motility’) consti-

tuted 36% of the annotated transcripts.

Association with arterial stiffeningAccording to the CRF status and aortic stiffness, there

was no interaction between the genes differentially

expressed. Moreover, we did not find any significant


on of smooth muscle cells (SMCs), cytoskeleton and apoptosis.

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Fig. 3

Gene and protein expression27%

Signaling/communication24%

Structure/motility12%

Metabolism9%

Cell division7%

Defense6%

Unclassified15%

36%

Classification of genes differentially expressed between chronic renalfailure (CRF) and non-CRF patients according to seven functionalcategories describes by Hwang et al. [23].

difference in PWV between CRF and non-CRF patients

of the two subpopulations (Table 1).

Protein analysisImmunohistochemical staining revealed an increased

amount of LUM and ODC1, two proteins corresponding

to the highest gene overexpression, in the vascular

smooth muscle cells (VSMCs) of CRF compared to

non-CRF aortic sections (Fig. 4). They also showed an

increased expression of osteocalcin and CAPZA1 (Fig. 4),

whereas OPG, dynein and MSN were not detected (data

not shown). Tissue preparations incubated without

primary antibody served as negative controls. Without

primary antibody, no immunoreactivity was detected in

these slides, which verified the specificity of the immu-

nostaining (data not shown).

In western blot analysis of aortic biopsies, the specific

antibodies directed against proteins of interest revealed

antigens that corresponded to the expected size (i.e.

78 kDa for MSN and 50 kDa for OPG). Moreover, the

levels of MSN appeared to be higher in CRF patients

compared to non-CRF patients (Fig. 5), consistent with

the 1.8-fold overexpression of the corresponding genes.

Interestingly, we observed an increased OPG expression

in aorta of CRF patients, whereas the gene expression level

was not significantly different between the two groups.

DiscussionThe main results of the present study are the following.

We demonstrated for the first time a specific gene expres-

sion profile in aortic tissues of patients with mild-to-

moderate CRF compared to patients with normal renal

function. Two genes had the highest overexpression in

CRF patients: LUM, involved in the regulation of col-


lagen fibrillogenesis; and ODC1, involved in polyamine

biosynthesis, SMC growth and proliferation. Immunohis-

tochemical staining revealed an increased amount of

LUM and ODC1 in the VSMCs of CRF compared to

non-CRF aortic sections. Among the differentially

expressed genes, an unexpectedly high number was

implicated in the migration of VSMCs and apoptosis.

Methodological featuresThe objective of the present study was to determine the

gene expression profile of aorta in CRF patients, and to

relate it to arterial stiffness, measured in vivo. This has

been little studied [16], as there are very few medical

indications for taking aortic biopsies from patients. The

main indication is coronary artery bypass surgery.

The microarray technique used in the present study

offered the possibility to hybridize two samples on the

same slide: a patient and the reference sample (donor). We

then normalized values of patients to values of the refer-

ence to eliminate the variability due to hybridization.

Moreover, the requirement of duplication of experiments

(use of swap) had improved the reproducibility of the

quantification; the specific signal associated with a spot

(representative of a gene) was defined by the geometrical

average of the specific intensities of this spot in direct and

swapped experiments. Although the number of samples

was small, we employed a rather strict set of filtering

criteria (Fig. 1). Transcripts were named ‘differentially

expressed’ only if they passed several independent filters.

Thus, the probability that these transcripts were associated

with CRF, only by chance, was very low.

Repeatability of findings obtained with microarray tech-

nique is often debated. We previously demonstrated the

robustness of our methodology [24]. Indeed, we observed

superimposed findings when using (a) either Affymetrix

chips [16] or the present chips supplied by CNRS UMR

6097 and (b) different sets of aortic samples (stiff and

distensible aortas). The percentage of differentially

expressed genes between stiff and distensible arteries

was similar (39 and 43%) when the two major gene

categories (cell structure/motility and cell signalling/com-

munication) were considered. Moreover, the fold changes

in the expression of genes of interest were similar when

the two sets of biopsies were compared. We can therefore

conclude that our methodology is robust enough to allow

us to draw conclusions in a small number of patients.

As the small size of the aortic biopsy did not allow us to

perform protein and gene expression analysis on the same

aortic sample, we performed immunohistochemistry and

western blot experiments on aortic biopsies originating

from CRF and non-CRF patients having the same charac-

teristics as those who had their aortic biopsy used for

microarray. Clinical characteristics were thus matched

between groups.


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Fig. 4

Immunohistochemical staining (red) of vascular smooth muscle cells (VSMCs) in human aortic sections of non-chronic renal failure (CRF) and CRFpatients. (a) CPAZA1 antibodies, (b) osteoprotegerin (OPG) antibodies, (c) lumican (LUM) antibodies and (d) ornithine decarboxylase (ODC1)antibodies; magnification is �40 for all images.

An advantage of aortic biopsies obtained during the

coronary artery bypass surgery was the lack of advanced

atherosclerosis. As shown in Fig. 4, sections of CRF

patients’ biopsies were histologically normal, without

atherosclerosis and overt calcifications. Thus, the over-

expression or underexpression of genes can be attributed

to the presence of CRF rather than to associated athero-

sclerosis. Although the lack of advanced atherosclerosis

may be at first surprising in these patients with extended

arterial disease, it may receive several explanations. The

coronary artery bypass graft was performed on the most

‘healthy’ part of the ascending aorta. The surgical pro-

cedure involved extensive palpation of the aortic wall

aiming at identifying regions in which the aortic wall is


free of calcifications, soft and distensible. In the present

aortic biopsy, LUM was found only in the media, and not

in the intima as expected in case of prominent athero-

sclerosis [25].

Interpretation of findingsIn the present study, we tested the hypothesis that in

mild-to-moderate CRF patients, the gene expression

profile of aortic tissues would include genes involved

in arterial calcifications. In patients with ESRD, vascular

calcification, especially that found in the media of large

arteries, leads to an increased arterial stiffening. Intimal

calcifications may occur independently of medial calcifi-

cation and vice versa, as a regulated biological process


C


Fig. 5

Western blot analysis of moesin (MSN) and osteoprotegerin (OPG)levels in the aorta of chronic renal failure (CRF) and non-CRF patients.Each lane corresponds to one patient. Values are mean�SEM.

following many of the cellular and molecular programs in

osteogenesis. Bone-related and bone-specific proteins are

expressed in intimal calcification, which occurs in macro-

phage-rich and lipid-rich atherosclerotic lesions [14,26].

Although our hypothesis could not be confirmed in a

statistically significant manner, a trend towards an upreg-

ulation of candidate genes involved in arterial calcifica-

tions and arterial enlargement was seen. In Monckeberg’s

sclerosis, Shanahan et al. [27] have demonstrated that

VSMC expression of MGP and osteonectin (SPARC) was

diminished, whereas expressions of ALPL, osteopontin

(SSP1), BGLAP and COL II were increased. In the

present study, although we observed an increased in

protein expression of two vascular calcification inhibitors

(OPG and osteocalcin), we did not find any significant

difference in the expression of these genes between CRF

patients and control patients. Nevertheless, we observed

a tendency for an overexpression of the markers of

osteogenic (ALPL, BGLAP, SPP1 and Msx2) and

chondrocytic (COL II) differentiations of the VSMCs

(Table 2) and an overexpression of genes implicated in

apoptosis (S100A8, DDX47 and CAPNS2). Human and

mouse genetic experiments have determined that blood

vessels normally express inhibitors of mineralization,

such as MGP and OPG. Interestingly, it has been demon-

strated that OPG protects endothelial cells from apopto-

sis [28], which can initiate VSMC calcification with

associated apoptotic body release [29]. Then a small

imbalance in favour of mineralization in CRF patients

can involve a downregulation of these inhibitors, con-

tributing to a loss of homeostatic inhibition of calcifica-

tion and, potentially, changes in VSMCs phenotype

[30,31]. In established calcification, there was evidence

for activation of both chondrocytic and osteogenic tran-

scriptional pathway in VSMCs [30]. Thus, our results

suggest that the process of vascular calcification was


initiated only to a limited extent, preventing a significant

difference between mild-to-moderate CRF and control

patients to be detected.

Two genes had the highest overexpression in CRF

patients: LUM, involved in the regulation of collagen

fibrillogenesis; and ODC1, involved in polyamine bio-

synthesis, SMC growth and proliferation. LUM was

three-fold upregulated both at the mRNA and protein

levels in CRF patients. This fibril-associated molecule is

a proteoglycan of the extracellular matrix, which is

involved in the regulation of collagen fibrillogenesis

[22]. LUM is implicated in the regulation of the pro-

gression of fibril assembly, though a series of steps lead-

ing to structurally and mechanically mature fibrils [32].

The collagen architecture is important for maintaining a

normal elasticity of blood vessels. Thus, modifications in

LUM production could affect collagen organization and

consequently, blood vessels elastic properties. Interest-

ingly, an overexpression of LUM mRNA has been

detected in kidney of rats subjected to chronic neonatal

unilateral ureteral obstruction [33] and in human diabetic

nephropathy [34]. LUM is abundantly expressed in the

murine bone matrix [35], in particular, during the stages

of mineralization and differentiation of murine calvarian

cells. Thus, upregulation of LUM may be functionally

important in the context of CRF-associated alterations

and possibly calcification of the aorta.

We also detected a significant 3.1-fold overexpression of

ODC1 in CRF patients. The ODC1 is involved in poly-

amine biosynthesis, required for sustaining their viability.

It has been demonstrated that pharmacological inhibition

of ODC1 activity inhibits rat aortic SMC proliferation

[36]. ODC1 expression is increased in distal tubules

of early diabetic rat kidneys, leading to hypertrophy of

kidney [37]. In contrast, pharmacological inhibition of

ODC1 leads to defects in kidney development in mice

[38]. ODC1 overexpression may suggest that arterial wall

hypertrophy of CRF patients [4] is, at least in part, due to

VSMCs proliferation.

We observed a significant upregulation of genes involved

in migration of VSMCs and cytoskeleton function, with

an increase in some of corresponding proteins in CRF

patients. Indeed, among the 101 genes differentially

expressed between CRF and non-CRF patients, an

unexpectedly high number of genes was implicated in

the regulation of actin polymerization (MSN, CAPZA1,

ARPC3, CAP1). Upregulation of these genes may lead to

disassembly of F-actin and phenotypic changes of

VSMCs towards migration. MSN, together with ezrin

and radixin, constitutes a gene family called ERM. These

proteins directly interact with actin filaments, under the

control of a Rho-dependent signalling pathway [39]. An

increased expression of MSN in VSMCs of the medial

wall could reflect a phenotypic change of VSMCs towards


C


migration, due to actin-filament (F-actin) disassembly

[40]. This is consistent with the fact that APOD is also

overexpressed (2.0-fold) in CRF patients. This major

component of HDL is associated with protease activity

and is required for breaking the cell–matrix attachments

in motile cells. An overproduction of APOD by VSMCs

may positively influence their migration [41]. As the actin

cytoskeleton is essential for maintaining the structural

and functional integrity of cells, our results suggest

that phenotypic changes towards migration and spatial

reorganization of aortic VSMCs occur at the early stage of

CRF. Indeed, in nephrectomized rats, VSMCs of the

aorta showed alteration of cell shape, orientation and

spatial arrangement of matrix, independently of blood

pressure [42]. That three genes involved in apoptosis

(S100A8, DDX47 and CAPNS2) were overexpressed in

CRF patients suggests an associated apoptosis. Both

VSMCs migration and apoptosis may contribute to the

arterial enlargement observed in CRF patients.

At the early stage of CRF, phenotypic changes of VSMCs

may favour the spatial reorganization of the arterial wall

towards lumen enlargement, in parallel with the initiation

of vascular calcification. Lumen enlargement, by loading

the stiff components of the arterial wall, in conjunction

with early vascular calcification could stiffen the arterial

wall.

In addition, the disorganization and abnormal distri-

bution of F-actin may play a role in the regulation of

vascular tone. In postischemic acute renal failure, the

disorganization of the actin cytoskeleton in VSMCs of

the renal vasculature may play a contributory role in the

increased vascular tone, impaired autoregulation of renal

blood flow and aberrant vascular reactivity [43].

Study limitationsSome study limitations should be discussed. First, the

present study included only a small number of patients

and aortic samples. This is due to the difficulty of

obtaining both aortic biopsies and noninvasive arterial

measurements before the bypass surgery, then collecting

enough aortic tissue despite the limited size of the

aortic biopsy, and finally extracting enough high-quality

RNA.

Second, by contrast to previous findings [16], aortic

stiffness was not increased in CRF compared to non-

CRF patients in the present study. This is very likely due

to the additional damage of the arterial wall in these

patients at high cardiovascular risk and scheduled for a

bypass surgery. Indeed, in patients with mild renal dys-

function and less numerous cardiovascular risk factors

[16], aortic stiffness was significantly elevated, although

mildly, by comparison with CRF hypertensives. The

patients included in the present study had a higher cf-

PWV than those of our former study [16], despite less


advanced renal dysfunction. Multivariate analyses of the

determinants of aortic stiffness [16,44] showed that GFR

had only a weak independent influence on aortic stiff-

ness. It is thus likely that CRF significantly influences

aortic stiffness only during the final stage of the disease,

whereas associated cardiovascular risk factors, leading to

atherosclerosis and coronary heart disease, are earlier

important determinants of aortic stiffness [1]. Finally,

because the entire group has very likely elevated aortic

stiffness as a result of their atherosclerosis, data of Table 1

suggest that there was widespread aortic disease in all

groups.

Third, antihypertensive agents have been implicated

in the control of vascular calcification and remodelling

[45]. Particularly, calcium channel blockers have been

reported to slow the progression of coronary calcification

in hypertensive patients [46]. In a novel model of isolated

systolic hypertension caused by arterial calcifications,

Dao et al. [47] and Essalihi et al. [48] have demonstrated

that a treatment with an ET-1 antagonist or an angio-

tensin II blocker prevents arterial calcifications and may

cause regression of vascular calcification when they are

established. It is unlikely that antihypertensive drugs

had masked any significant increase in expression of

genes involved in vascular calcification, as only two

and three patients among CRF patients and controls,

respectively, had antihypertensive medications. Never-

theless, it should be kept in mind that lipid-lowering

therapy and antihypertensive drugs, which can delay

the progression or lead to regression of atherosclerotic

disease, can have an impact on the genetic translation of

the different factors involved in these biochemical

processes.

In conclusion, these results show that patients at an early

stage of CRF have a specific gene expression profile of

aortic tissue and suggest that genes implicated in collagen

fibrillogenesis and VSMCs migration and proliferation

may play a role, particularly LUM and ODC1.

AcknowledgementThe present study was supported by grants from the

‘Fondation de France’, INSERM (Institut National de la

Sante et de la Recherche Medicale) and ANR-05-PCOD-

004-0 (Agence Nationale de la Recherche).

There are no conflicts of interest.

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