ARTHRITIS & RHEUMATISMVol. 63, No. 11, November 2011, pp 3552–3562DOI 10.1002/art.30536© 2011, American College of Rheumatology

Insights Into the Pathogenesis of Systemic SclerosisBased on the Gene Expression Profile of

Progenitor-Derived Endothelial Cells

Jerome Avouac,1 Nicolas Cagnard,2 Jorg H. Distler,3 Yoland Schoindre,2 Barbara Ruiz,2

Pierre Olivier Couraud,2 Georges Uzan,4 Catherine Boileau,3

Gilles Chiocchia,2 and Yannick Allanore1

Objective. To determine the gene expression pro-file of endothelial cells derived from the endothelialprogenitor cells (EPCs) of patients with systemic scle-rosis (SSc).

Methods. Microarray experiments were per-formed on Affymetrix GeneChip Human Exon 1.0 STArrays in unstimulated and hypoxia-stimulated EPC-derived cells from patients with SSc and control sub-jects. Followup of the raised hypotheses was performedex vivo by immunohistochemical analysis of skin tissue.

Results. Signals from 92 probe sets and 188 probesets were different in unstimulated and hypoxia-stimulated cells, respectively, from patients with SSccompared with controls. Within the largest groups ofgenes related to cell–cell interaction and vascular re-modeling, down-regulation of tumor necrosis factorligand superfamily member 10 (TNFSF10) and homeo-box A9 (HOX-A9) was confirmed by real-time polymer-

ase chain reaction and Western blots in EPC-derivedcells and by immunohistochemistry in SSc skin tissue.Signals from 221 and 307 probe sets were different inunstimulated and hypoxia-stimulated cells, respectively,from patients with diffuse cutaneous SSc compared withpatients with limited cutaneous SSc. Within the largestgroup of genes related to the inflammatory response,differential expression of TNF�-induced protein 3 andprostaglandin-endoperoxide synthase 2 was observed inEPC-derived cells and skin tissue from patients withSSc.

Conclusion. Our data revealed important geneexpression changes in EPC-derived endothelial cellsfrom patients with SSc, characterized by a proadhesive,proinflammatory, and activated phenotype. Differentialexpression in lesional SSc skin tissue of new targets,such as TNF family members and HOX-A9, may con-tribute to the pathogenesis of SSc and deserves morein-depth exploration.

Systemic sclerosis (SSc) is a severe connectivetissue disorder of unknown origin that affects the skinand internal organs (1). Microvascular alterations arekey features of the disease, with outcome depending onthe extent and severity of vascular lesions (2,3). Studieswith animal models have shown that endothelial cellapoptosis could be a primary event in the pathogenesisof SSc (4–7). Endothelial cell damage and apoptosisresult in disorganization of the capillary architecture andloss of capillaries, which are constant at all stages of thedisease (8). This decreased capillary density results ininsufficient blood flow and reduces the supply of oxygen,which leads to tissue hypoxia.

Despite the importance of endothelial cells in thepathogenesis of SSc, rigorous analysis of these cells is

Supported by the Association des Sclerodermiques de France,Groupe Francais de Recherche sur la Sclerodermie (MutuelleAMPLI), INSERM, Agence Nationale pour la Recherche (grantR07094KS), and the Fondation Arthritis. Dr. Allanore is recipient ofan investigator-initiated research grant from Pfizer.

1Jerome Avouac, MD, PhD, Yannick Allanore, MD, PhD:Universite Paris Descartes and Hôpital Cochin, AP-HP, and INSERMU1016, Cochin Institut, Paris, France; 2Nicolas Cagnard, MSc, YolandSchoindre, MD, Barbara Ruiz, Pierre Olivier Couraud, PhD, GillesChiocchia, PhD: INSERM U1016, Cochin Institut, Paris, France; 3JorgH. Distler, MD, Catherine Boileau, PharmD, PhD: U.V.S.Q. Univer-sity, Ambroise Hospital, APHP, Boulogne, France; 4Georges Uzan,PhD: INSERM U972, Hôpital Paul Brousse, Villejuif, France.

Dr. Distler has received consulting fees, speaking fees, and/orhonoraria from Actelion, Pfizer, Bayer, Schering, Encysive, and Glaxo-SmithKline (less than $10,000 each).

Address correspondence to Yannick Allanore, MD, PhD,Hopital Cochin, Service de Rhumatologie A, 7 rue du Faubourg St.Jacques, 75014 Paris, France. E-mail: yannick.allanore@cch.aphp.fr.

Submitted for publication November 25, 2010; accepted inrevised form June 28, 2011.

3552

lacking because of the difficulty of accessing humantissues (9). We recently developed a noninvasive methodto isolate in cell culture endothelial cells derived fromcirculating endothelial progenitor cells (EPCs); this ap-proach was emphasized in recent recommendationsfrom the European League Against Rheumatism(EULAR)/EULAR Scleroderma Trial and Researchgroup (10–12). Late-outgrowth EPC-derived endothelialcells may represent an original and valuable cellularmodel to investigate endothelial biology in SSc, becausethey have the phenotype of genuine endothelial cells,display robust proliferative potential, exhibit in vitroangiogenic properties, and have the capacity to consti-tute and orchestrate vascular remodeling in vivo (13–15).

Considering the endothelial cell dysfunction anddamage that occur in SSc, we hypothesized that geneexpression analysis of EPC-derived endothelial cellsfrom patients with SSc may offer a novel approach toidentify differentially expressed genes, raising new po-tential targets and pathways of interest. To validate thishypothesis, we investigated the gene expression pro-file in late-outgrowth EPC-derived endothelial cellsfrom patients with SSc and control subjects, using a2-stage strategy including 1) a comprehensive micro-array analysis, and 2) followup of the raised hypotheses,using messenger RNA (mRNA) and protein lysatesfrom EPC-derived endothelial cells and skin tissue.

PATIENTS AND METHODS

Patients. Late-outgrowth EPC-derived cells were ob-tained from the peripheral blood of 10 patients (80% of whomwere female) who met the American College of Rheumatologycriteria for SSc (16). The mean � SD age of the patients was61 � 12 years, and the mean � SD disease duration was 9 �9 years. Five patients (50%) had the limited cutaneous subtypeof SSc (lcSSc), and none had ever received immunosuppressivedrugs. All patients were treated with calcium channel blockers.Control EPC-derived cells were obtained from 5 healthywomen, with a mean � SD age of 63 � 9 years. None of thesubjects included in this study had systemic arterial hyperten-sion, diabetes mellitus, dyslipidemia, or a history of smoking.For immunohistochemistry experiments, skin sections wereobtained from an extended population of 20 patients with SSc(85% female, mean � SD age 60 � 10 years, mean � SDdisease duration 10 � 9 years, 55% lcSSc) and 10 matchedcontrol subjects. Local ethics review committees approved allexperiments, and all patients and control subjects providedwritten informed consent.

Late-outgrowth EPC isolation. EPC isolation wasperformed on a 50-ml heparinized venous blood sample ob-tained from the forearm. Samples from hospitalized patientswere obtained in the morning, at rest, during routine analysis.Patient and control samples were immediately transported tothe laboratory for testing.

We used a previously described culture method thatwas suitable for obtaining and expanding late-outgrowth EPC-derived colonies (10,14). Briefly, the blood mononuclear cellfraction was collected by Ficoll density-gradient centrifugationand was resuspended in complete endothelial cell growthmedium 2 (EGM-2; Lonza). Cells were then seeded ontoseparate wells of a 12-well tissue culture plate precoated withtype I collagen (rat tail; BD Biosciences) and stored in anatmosphere of 5% CO2 at 37°C in a humidified incubator.After 24 hours of culture, nonadherent cells and debris wereaspirated, adherent cells were washed once with 1� phosphatebuffered saline (PBS), and complete EGM-2 was added toeach well. The medium was changed daily for 7 days and thenevery other day until the first passage. Colonies of endothelialcells appeared between 8 days and 26 days of culture and wereidentified as well-circumscribed monolayers of cells with acobblestone appearance. After the third passage, phenotypingof EPC-derived cells was performed by flow cytometry, aspreviously described (13). After confirmation of the endothe-lial phenotype, cells were suspended in fetal bovine serumsupplemented with 20% DMSO, frozen in liquid nitrogen, andstored until used.

Hypoxia treatment. For hypoxia experiments, 1 � 106

cells were seeded into 100-mm dishes. After 3 days, when cellswere subconfluent, the medium was changed, and cells wereexposed to hypoxia for 6 hours (in an atmosphere of 1% O2and 5% CO2 at 37°C in a humidified incubator).

Microarray analysis. All 15 samples were defrosted, cul-tured, extracted, and hybridized at the same time. Affymetrixmicroarray technology was used to analyze gene expressionlevels. Labeling and microarray processing were performedaccording to the manufacturer’s protocol.

RNA preparation. Total RNA was extracted fromcultured cells in buffer RLT (Qiagen RNeasy kit) and treatedwith DNase I to eliminate genomic DNA contamination. Theintegrity and purity of total RNA and complementary RNA(cRNA) were analyzed twice using the Agilent 2100 Bioana-lyzer and the RNA 6000 LabChip kit. Only total RNA with a28S:18S ratio of �1.9 was used. Complementary RNA concen-trations were measured using a NanoDrop spectrophotometer.

Complementary RNA synthesis and probe array hy-bridization. Complementary RNA synthesis was performedwith 3 �g of total RNA, using the GeneChip Expression 3�Amplification One-Cycle Target Labeling and Control Re-agents kit, hybridized onto the GeneChip Human Exon 1.0 STArray (Affymetrix) according to the manufacturer’s protocol(GeneChip Whole Transcript Sense Target Labeling AssayManual). Briefly, the majority of the ribosomal RNA (rRNA)was removed from the total RNA samples for increasedsensitivity, using the RiboMinus Human/Mouse Transcrip-tome Isolation Kit (Invitrogen). Next, RNA depleted fromrRNA was reverse transcribed using a T7-oligo(dT) promoterprimer in the first-strand cDNA synthesis reaction. Follow-ing RNase H–mediated second-strand cDNA synthesis,the double-stranded cDNA was purified and served as a tem-plate in the subsequent in vitro transcription (IVT). TheIVT reaction was carried out in the presence of T7 RNApolymerase and a biotinylated nucleotide analog/ribonucleotidemix for cRNA amplification and biotin labeling. The bio-tinylated cRNA targets were then cleaned up and frag-

A GENE EXPRESSION SIGNATURE FOR SYSTEMIC SCLEROSIS 3553

mented. Fragmentation was evaluated on a bioanalyzer.Selected fragmented cRNA were hybridized to GeneChipexpression arrays. After washing and staining using theAffymetrix Fluidics Station 450, the probe arrays were scannedinto the Affymetrix GeneChip Scanner 3000 7G system.

Data processing and analysis. The microarray filescontaining the whole database have been uploaded to MIAME(Minimum Information About Microarray Experiment; http://www.ebi.ac.uk/arrayexpress/) (additional information is avail-able from the corresponding author). Fluorescence data wereimported into Affymetrix Expression Console software andR/Bioconductor software. Gene expression levels were calcu-lated using the robust multiarray average algorithm at genelevel in Expression Console, and flags were computed using acustom algorithm within R. To limit potentially biased mea-surement (background or saturating), all probes for which thenormalized intensity measures were outside of a confidenceinterval were flagged 0. The confidence interval was �2 SDfrom the mean intensities of each chip.

Three probe lists were used for each comparisonaccording to flagged measurement in the relevant chips. The“PP” list comprises probes flagged only as “present” for allchips involved in the comparison. The “P50” list was created byfiltering probes flagged as “present” for at least half of thechips. The “All” list comprises all probes, without any filtering.

Each comparison was performed in 2 steps. The firststep, unsupervised analysis by hierarchical clustering using the3 probe lists, allowed an estimation of intragroup homogeneityby revealing natural groups. Next, group comparisons wereperformed using Student’s t-test in GeneSpring. We filteredthe resulting P values at 5% with a fold change �1.5. Bonfer-roni adjustment was applied to estimate the family-wise errorrate, and the Benjamini and Hochberg method was used toestimate the false-discovery rate. Because these correctionmethods were very stringent and precluded the identificationof some genes of interest, comparisons were also performedwithout correction. Cluster analysis was performed by hierar-chical clustering, using Spearman’s correlation and an averagelinkage algorithm. Data were subsequently submitted to theIngenuity Pathway Analysis database to reveal relevant bio-logic processes. We also conducted a literature search usingNational Center for Biotechnology Information databases,including PubMed, for all significantly overexpressed or un-derexpressed gene transcripts to determine their biologicfunction.

Validation of genes identified at the mRNA and pro-tein levels. Differentially expressed genes belonging to themain relevant biologic processes were selected on the basisof their odds ratio (�0.5 for down-regulated genes or �2 forup-regulated genes) for further validation by quantitative re-verse transcription–polymerase chain reaction (RT-PCR) andWestern blot analysis. If confirmation was obtained, proteinexpression was investigated by immunohistochemistry in con-trol and SSc lesional skin tissue.

Gene expression assays under basal and hypoxicconditions. For quantitative RT-PCR, the TaqMan methodwas performed as previously described (13). We used primersequences (inventoried by Applied Biosystems) for tumornecrosis factor ligand superfamily member 10 (TNFSF10;Hs00921974-m1), TNFSF18 (Hs00183225-m1), Kit ligand(Hs00241497-m1), homeobox A9 (HOX-A9; Hs00365956-m1),

prostaglandin-endoperoxide synthase 2 (PTGS-2; Hs00153133-m1), and TNF�-induced protein 3 (TNFAIP3; Hs00234713-m1). Hypoxanthine guanine phosphoribosyltransferase(Hs99999909-m1) was used as a housekeeping control tonormalize the amounts of cDNA within each sample. All PCRswere performed in triplicate. Differences were calculated usingthe Ct and comparative Ct methods for relative quantification.Results are expressed as the relative expression (%) of mRNAlevels.

Western blot analysis. Western blotting was performedas previously described (13). Immunoblots were incubatedovernight at 4°C with mouse anti-human monoclonal anti-bodies against PTGS-2 (Abcam) at a dilution of 1:2,500, rabbitanti-human monoclonal antibodies against TNFAIP3 (Abcam)at a dilution of 1:5,000, goat anti-human polyclonal antibodiesagainst HOX-A9 (Santa Cruz Biotechnology) at a dilution of1:500, or mouse anti-human monoclonal antibodies againstTNFSF10 (Santa Cruz Biotechnology) at a dilution of 1:200.Horseradish peroxidase–conjugated goat anti-rabbit, rabbitanti-mouse, or rabbit anti-goat antibodies (Dako) were used assecondary antibodies at a dilution of 1:10,000 for 1 hour.

Immunohistochemical analysis. Skin sections (7 �m)were mounted on SuperFrost slides, following the protocol ofthe hospital pathology laboratory. Sections were deparaf-finized and rehydrated, and antigen retrieval was performed.Slide-mounted sections were heated in a microwave oven at700 watts, twice for 10 minutes in 10 mmoles/liter sodiumcitrate buffer (pH 6.0). Tissue sections were allowed to cool atroom temperature. The sections were then blocked with 5%normal horse serum in 1� PBS–2% bovine serum albumin(BSA) for 1 hour and incubated overnight at 4°C with 2% BSAin PBS and rabbit anti-human TNFAIP3, PTGS-2, andTNFSF10 polyclonal antibodies (dilutions of 1:250, 1:500, and1:500, respectively; Abcam) and goat anti-human HOX-A9polyclonal antibody (1:100 dilution; Santa Cruz Biotechnol-ogy). Next, endogenous peroxidase was inactivated with 3%H2O2 in PBS for 10 minutes. Sections were washed in PBS andincubated for 1 hour with 2% BSA in PBS and goat anti-rabbitor rabbit anti-goat IgG antibodies (1:200 dilution; Dako). Thissecondary antibody was detected with the DAB� PeroxidaseSubstrate Kit (Dako). Immunoreactivity was quantified usingcomputer-assisted color image analysis (Nikon Eclipse 80imicroscope and Sony DSP 3CCD camera).

Two investigators blinded to the diagnosis evaluated 6randomly selected high-power fields for each section of 3 skinbiopsy samples per patient or control subject. Semiquantitativeevaluation of the immunohistochemical reactions was per-formed using a modified version of the score previouslydescribed by Zhang et al, based on the percentage of positivecells and the intensity of immunoreactivity (17). The averageimmunoreactivity was determined for each patient and controlsubject, based on the results for the 3 slides. The averageimmunoreactivity was calculated for each SSc and controlpopulation. Scores were compared, and a consensus readingwas performed in case of discrepancies.

Statistical analysis. All data are presented as themean � SD, unless stated otherwise. Comparisons betweenany 2 groups were performed using unpaired t-tests for nor-mally distributed data and the nonparametric Mann-Whitneytest for non-normally distributed data. P values less than 0.05were considered significant.

3554 AVOUAC ET AL

Figure 1. Cluster analyses according to gene expression profiles in unstimulated and hypoxia-stimulated endothelial progenitor cell (EPC)–derivedcells from patients with systemic sclerosis (SSc) compared with controls. A and C, Unsupervised analyses performed on the “All” list of probesaccording to a P value of �0.05 without correction and a fold change of �1.5, showing correct classification of 90% of patients with SSc and 100%of controls under basal conditions (A) and 100% of patients and 80% of controls under hypoxic conditions (C). B and D, Histograms showing the10 main relevant biologic processes differentially expressed in unstimulated and hypoxia-stimulated EPC-derived endothelial cells, respectively, frompatients with SSc and controls. Bars show the significance (�log10 P) of each biologic process (threshold set at P � 0.05).

Table 1. Differentially expressed genes in the most relevant biologic processes identified by IngenuityPathway Analysis

Biologic processNo. ofgenes

Underexpressedgenes

Oddsratio

Overexpressedgenes

Oddsratio

Basal conditionsCell–cell signaling and interaction 6 PCDH10 0.41 TNFSF18 2.56

TNFSF10 0.32 KITLG 2.34CD200 0.50 PCSK1 1.89

Cellular growth and proliferation 2 TNFSF10 0.32 KITL 2.34Hematologic system development

and function5 TNFSF10 0.32 KITL 2.34

BST1 1.62HDAC9 1.85CIAPIN1 1.81

Humoral immune response 5 TNFSF10 0.32 KITL 2.34CD200 0.50 BST1 1.62KLRG1 0.62

Hypoxic conditionsGene expression 4 HOXA2 0.62

HOXA5 0.64HOXA9 0.35HOXA11 0.62

Organism development 5 HOXA2 0.62HOXA5 0.64HOXA9 0.35HOXA11 0.62FRAS1 0.65

A GENE EXPRESSION SIGNATURE FOR SYSTEMIC SCLEROSIS 3555

RESULTS

Gene expression in EPC-derived endothelialcells. Unsupervised analyses by hierarchical clusteringusing the 3 probe lists did not allow correct segregationbetween patients and controls for both unstimulated andhypoxia-stimulated EPC-derived cells (additional infor-mation is available from the corresponding author). Thefollowing results were derived from an unsupervisedanalysis performed on the “All” list of probes accordingto a P value less than 0.05 without correction and a foldchange of �1.5.

Gene expression changes in unstimulated EPC-derived endothelial cells from SSc patients compared withhealthy controls. Analyses performed under basal condi-tions on cells from 10 patients with SSc and 5 controlsubjects allowed a correct classification for 90% of SScpatients and 100% of controls (Figure 1A). We detected33 genes (92 probe sets) that were differentially regu-

lated in unstimulated EPC-derived cells from patientswith SSc compared with controls (additional informationis available from the corresponding author). Four mainbiologic processes were identified after Ingenuity Path-way Analysis (Figure 1B and Table 1). Among these,differential expression of 2 members of the TNF super-family was of particular interest. We observed down-regulation of TNFSF10 in EPC-derived cells from pa-tients with SSc. The best-characterized biologic activityof TNFSF10 is to induce apoptotic cell death in a varietyof neoplastic cells. TNFSF10 also counteracts the pro-adhesive activity of inflammatory cytokines in endothe-lial cells, preventing the interaction between peripheralblood–derived monocytes and endothelial cells (18). InEPC-derived cells from patients with SSc, we also ob-served up-regulation of both TNFSF18, which is animportant factor for interaction between T lymphocytesand endothelial cells, and Kit ligand, a factor that

Figure 2. Cluster analyses according to gene expression profiles in hypoxia-stimulated EPC-derived cells from patients with limited cutaneous SSc(lcSSc) and patients with diffuse cutaneous SSc (dcSSc). A and C, Unsupervised analyses performed on the “All” list of probes according to a P valueof �0.05 without correction and a fold change of �1.5, showing correct classification of 100% of patients with lcSSc (patients 1–5) or dcSSc (patients6–10) under both basal (A) and hypoxic (C) conditions. B and D, Histograms showing the 10 main relevant biologic processes differentially expressedin unstimulated and hypoxia-stimulated EPC-derived endothelial cells from patients with lcSSc (B) and those with dcSSc (D). Bars show thesignificance (�log10 P) of each biologic process (threshold set at P � 0.05). See Figure 1 for other definitions.

3556 AVOUAC ET AL

stimulates the growth and mobilization of hemangio-blasts (19–21).

Gene expression changes in hypoxia-stimulatedEPC-derived endothelial cells from SSc patients comparedwith healthy controls. Unsupervised analyses performedon hypoxia-stimulated cells from 10 patients with SScand 5 controls allowed correct classification of 100%of SSc patients and 80% of controls (Figure 1C). Wedetected 53 genes (188 probe sets) that were differen-tially expressed in EPC-derived cells from patients withSSc compared with controls. Eight of these genes werethe same as those detected in unstimulated EPC-derivedcells (additional information is available from the corre-sponding author). The 2 main relevant biologic pro-cesses identified involved the homeobox genes, whichare known to regulate angiogenesis and vascular remod-eling (Figure 1D and Table 1) (22,23). In particular,HOXA9, a factor essential for the migration, tube-forming capacity, and activation of mature endothelialcells, was down-regulated in EPC-derived cells frompatients with SSc (24).

Gene expression changes in unstimulated andhypoxia-stimulated EPC-derived endothelial cells from pa-tients with dcSSc compared with those with lcSSc. Un-supervised analyses performed on unstimulated andhypoxia-stimulated EPC-derived cells from 5 patientswith lcSSc and 5 patients with dcSSc allowed correctclassification of 100% of the patients (Figures 2A andC). We detected 75 genes (221 probe sets) and 179 genes(307 probe sets) that were differentially expressed inunstimulated and hypoxia-stimulated EPC-derived cells,respectively, from patients with SSc (additional informa-tion is available from the corresponding author). Asignificant part of the genes identified in the meanrelevant biologic processes were involved in inflamma-tory and immune responses (Table 2 and Figures 2B andD). In addition to the up-regulation of interleukin-6(IL6) and VCAM1, which are known to contribute to thepathogenesis of SSc, we observed strong up-regulationof PTGS2 under both basal and hypoxic conditions andin TNFAIP3 after exposure to hypoxia (25–28). PTGS-2acts as a major mediator of inflammation, and TNFAIP3

Table 2. Gene expression changes in EPC-derived endothelial cells from patients with dcSSc comparedwith patients with lcSSc*

Biologic processNo. ofgenes

Underexpressedgenes in patients

with dcSScOddsratio

Overexpressedgenes in patients

with dcSScOddsratio

Basal conditionsOrgan morphology 7 FZD3 0.64 VCAM1 1.86

IL6 1.85PTGS2 2.95LIF 1.57CDKNI1A 1.50PRKG1 1.91

Reproductive system developmentand function

4 IL6 1.85PTGS2 2.95LIF 1.57CDKNI1A 1.50

Immune response 3 SELP 0.59 VCAM1 1.86PTGS2 2.95IL6 1.85

Hypoxic conditionsLipid metabolism 4 ALDH1A1 0.54 PTGS2 2.74

PTGIS 1.54SGMS2 1.51

Immune response 5 TNFAIP3 2.51PTGS2 2.74CD200 1.80CCR1 1.66IFNGR1 1.69TIMP3 1.51

* EPC � endothelial progenitor cell; dcSSc � diffuse cutaneous systemic sclerosis; lcSSc � limitedcutaneous SSc.

A GENE EXPRESSION SIGNATURE FOR SYSTEMIC SCLEROSIS 3557

is a negative regulator of NF-�B activation and aninhibitor of TNF�-mediated apoptosis, limiting inflam-mation by terminating the TNF� and NF-�B responses.

Followup of the hypotheses raised. MessengerRNA and protein expression of new targets in EPC-derivedendothelial cells. Consistent with the results of the micro-array analysis, TNFSF10 mRNA levels were decreased

in unstimulated and hypoxia-stimulated EPC-derivedendothelial cells from patients with SSc (mean � SEM51 � 12% [P � 0.02] and 59 � 9% [P � 0.02], re-spectively) compared with controls (Figure 3A).TNFSF10 protein expression was also reduced underbasal and hypoxic conditions, by 54 � 5% (P � 0.03) and57 � 4% (P � 0.03), respectively (Figure 3B). HOXA9

Figure 3. TNFSF10, HOX-A9, PTGS-2, and TNFAIP3 mRNA and protein levels as determined by quantitative reverse transcription–polymerasechain reaction and Western blot analyses in late-outgrowth EPC-derived cells. A and B, Decreased TNFSF10 mRNA and protein levels wereobserved in unstimulated and hypoxia-stimulated EPC-derived cells from patients with SSc. C and D, HOXA9 mRNA and protein levels weresignificantly reduced following hypoxia stimulation in EPC-derived endothelial cells from patients with SSc. E and F, PTGS2 mRNA and proteinlevels were significantly increased in unstimulated and hypoxia-stimulated EPC-derived cells from patients with diffuse cutaneous SSc (dcSSc)compared with controls and patients with limited cutaneous SSc (lcSSc). G and H, Patients with dcSSc had increased TNFAIP3 mRNA and proteinlevels in hypoxia-stimulated EPC-derived cells compared with patients with lcSSc. Western blots were performed in 10 SSc patients and 5 controls.Values are the mean � SEM. � � P � 0.05 versus controls; § � P � 0.05 versus limited SSc. See Figure 1 for other definitions.

3558 AVOUAC ET AL

mRNA and protein levels were decreased in hypoxia-stimulated EPC-derived cells from patients with SSc, by81 � 13% (P � 0.01) and 46 � 7% (P � 0.008),respectively (Figures 3C and D). TNFSF10 and HOXA9mRNA and protein levels were not significantly differ-ent between the lcSSc and dcSSc subgroups (data notshown). The up-regulation of TNFSF18 and KITLG wasnot confirmed at either the mRNA or the protein level(data not shown).

PTGS2 mRNA levels were increased by amean � SEM of 130 � 18% (P � 0.04) under basalconditions and 94 � 26% (P � 0.04) following hypoxiastimulation in patients with dcSSc compared with con-trols. PTGS2 mRNA levels were also increased, by130 � 25% (P � 0.04) under basal conditions and by112 � 28% (P � 0.03) following hypoxia stimulation inpatients with dcSSc compared with patients with lcSSc(Figure 3E). Similar results were observed at the proteinlevel (Figure 3F). A trend for increased expression ofPTGS-2 was also observed in the whole sample of SScpatients compared with controls, but this difference didnot reach significance.

TNFAIP3 mRNA and protein levels were in-creased by a mean � SEM of 54 � 12% (P � 0.03) and48 � 14% (P � 0.01), respectively, in hypoxia-stimulatedEPC-derived cells from patients with dcSSc comparedwith patients with lcSSc (Figures 3G and H). We alsoobserved a trend toward significance for decreasedmRNA expression of TNFAIP3 in the whole sample ofSSc patients compared with controls.

Tissue expression of identified new targets. Theexpression of TNFSF10, HOX-A9, and TNFAIP3 wassignificantly reduced in the skin of patients with SSc(Figures 4A–D, G, and H). TNFSF10, HOX-A9, andTNFAIP3 proteins were detected ex vivo by immuno-histochemistry in 70%, 40%, and 60% of SSc patientsand in 100%, 80%, and 100% of controls, respectively.According to our semiquantitative score, the intensityof immunoreactivity for TNFSF10, HOX-A9, andTNFAIP3 was decreased in SSc endothelial cells, inflam-matory cells, and fibroblasts compared with controls. Inaddition, the percentage of cells stained for TNFSF10and TNFAIP3 was reduced by a mean � SEM of 55 �7% (P � 0.02) and 62 � 5% (P � 0.02), respectively.Nuclear accumulation of HOX-A9 was also decreasedby 41 � 3% (P � 0.04) in patients with SSc.

Expression of PTGS-2 was up-regulated in skinbiopsy specimens obtained from patients with SSc(Figures 4E and F). PTGS-2 was detected in 100% ofpatients with SSc and in 40% of controls. The intensity

Figure 4. Expression of TNFSF10, HOX-A9, PTGS-2, and TNFAIP3in skin samples from patients with systemic sclerosis (SSc) and controlsubjects. Skin expression of the targets was assessed ex vivo byimmunohistochemistry in an extended population of 20 patients withSSc and 10 control subjects. Overall immunoreactivity was scored in a4-tier scale system based on the percentage of positive cells and theintensity of immunoreactivity. Immunoreactivity was scored as follows:� � negative result; � � weak immunoreactivity regardless of thepercentage of the cells being positive; �� � moderate immunoreac-tivity in �75% or strong immunoreactivity in �25% of the cells; and��� � moderate immunoreactivity in �75% or strong immunore-activity in �25% of the cells. The expression of TNFSF10 (A and B),HOX-A9 (C and D), and TNFAIP3 (G and H) was significantlyreduced in the skin of SSc patients compared with controls. PositiveTNFSF10 staining was observed mainly in endothelial cells, fibroblasts,and inflammatory cells (A1, A2, B1, and B2). Positive HOXA9staining was observed preferentially in endothelial cells (C1 and D1),and positive TNFAIP3 staining was detected in endothelial cells andinflammatory cells (G1, G2, H1, and H2). The expression of PTGS-2was up-regulated in SSc skin biopsy specimens (E and F). Positivestaining was localized mainly in endothelial and inflammatory cells(E1, E2, F1, and F2). Arrows indicate cells positive for TNFSF10,HOXA9, PTGS2, and TNFAIP3, respectively.

A GENE EXPRESSION SIGNATURE FOR SYSTEMIC SCLEROSIS 3559

of immunoreactivity was increased mainly in endothelialand inflammatory cells. The percentage of positive cellswas increased by a mean � SEM of 69 � 5% (P � 0.01).No significant difference in the staining intensity or thenumber of stained cells was observed between patientswith lcSSc and those with dcSSc.

DISCUSSION

This study is the first to evaluate the gene expres-sion profile of late-outgrowth EPC-derived endothelialcells, which represent a new and pertinent cellularmodel, because these cells have the genuine phenotypeof endothelial cells and display angiogenic properties invitro and in vivo. Furthermore, the raised hypotheseswere followed up at the mRNA and protein levels, usingboth EPC-derived endothelial cells and skin tissue.

An original feature of our study is the assessmentof gene expression of EPC-derived cells under hypoxicconditions. Identification of new targets in hypoxia-stimulated endothelial cells might be particularly rele-vant for SSc, because severe tissue hypoxia is a hallmarkof this disease and contributes directly to its progression(29). Our results showed that hypoxia modulates thegene expression profile of late-outgrowth EPC-derivedendothelial cells obtained from patients with SSc. Thedifferent profiles identified under hypoxic conditionsmay account for a proadhesive, activated, and pro-inflammatory phenotype of these cells in SSc, which maypromote the development of vasculopathy.

Apoptosis of endothelial cells and perivascularinflammatory cell infiltration are 2 of the earliest phe-nomena involved in the pathogenesis of SSc. Adhesionand interaction between activated endothelial cells andinflammatory cells are crucial to enable the transmigra-tion of inflammatory cells through the endothelium. Thedifferential expression of a large group of genes involvedin cell–cell interaction in EPC-derived endothelial cellsfrom patients with SSc suggests a proadhesive profileof these cells. Consistent with these findings, anothermicroarray study performed on circulating CD45� cellsobtained from the peripheral blood of patients with SScrevealed a dysregulation of the genes involved in celladhesion (30).

Within the group of genes involved in cell–cellinteraction, we observed a down-regulation of TNFSF10(TRAIL), which was further confirmed at the mRNAand protein levels under both basal and hypoxic condi-tions. Moreover, reduced expression of TNFSF10 wasobserved in the skin of patients with SSc, especially inendothelial and inflammatory cells. Because recent data

have shown that TNFSF10 plays an important role inmodulating leukocyte/endothelial cell adhesion, our re-sults suggest that down-regulation of TNFSF10 mightpromote the adhesion of circulating mononuclear cellsto activated endothelial cells in SSc (18). These prelim-inary findings will require further confirmation withfunctional analyses.

Because vascular alterations are a key event inthe pathogenesis of SSc, much attention has been givento cytokines, chemokines, and growth factors that regu-late the activation of endothelial cells (31–33). Ourmicroarray analysis identified a family of transcriptionfactors, the homeobox genes, that were down-regulatedunder hypoxic conditions in EPC-derived cells frompatients with SSc. Within this group of genes, we con-firmed the down-regulation of HOXA9 at the mRNAand protein levels. Moreover, reduced expression ofHOX-A9 was also observed ex vivo in the skin of SScpatients, especially in endothelial cells. Although thedirect effects of decreased expression of HOX-A9 havenot yet been addressed in SSc, indirect evidence suggeststhat its down-regulation might promote activation andabnormal differentiation of endothelial cells. HOX-A9has been shown to negatively regulate endothelial acti-vation by suppressing adhesion molecule expression inresponse to TNF� and inhibiting NF-�B–dependenttranscription (24). Moreover, recent knockdown andoverexpression studies have revealed that HOX-A9 actsas a master switch to regulate the expression of proto-typical endothelial-committed genes and mediates theshear stress–induced maturation of endothelial cells (22).

Gene expression changes revealed a proinflam-matory profile of late-outgrowth EPC-derived endo-thelial cells from patients with dcSSc. We identified 2new targets, PTGS-2 and TNFAIP3, that play an impor-tant role in the inflammation process, which passedthrough preliminary validation by quantitative RT-PCRand Western blot analysis. Moreover, differential ex-pression of these factors has been observed in thelesional skin of patients with SSc. PTGS-2, also knownas cyclooxygenase, is a key inducible enzyme in prosta-glandin biosynthesis, the expression of which is inducedby a wide variety of extracellular stimuli as part of theinflammatory response (e.g., in connective tissue dis-eases) (34). Enhanced expression of PTGS-2 was ob-served in fibroblasts obtained from the lesional skin ofpatients with SSc and contributes to the IL-1�—dependent up-regulation of prostaglandin E2. Consis-tent with this observation, we observed up-regulation ofPTGS-2 in skin biopsy specimens obtained from patientswith dcSSc or lcSSc, with strong immunoreactivity in

3560 AVOUAC ET AL

endothelial and inflammatory cells. We also observedincreased mRNA and protein levels of PTGS2 in EPC-derived endothelial cells from patients with dcSSc, sug-gesting that PTGS-2 might also contribute to the devel-opment of SSc vasculopathy in this subgroup of patients.

Dysregulation of TNFAIP3 expression in EPC-derived cells and skin biopsy specimens from patientswith SSc must be related to recent genetic data, whichhave identified TNFAIP3 as a new susceptibility factorfor SSc (35). TNFAIP3 is a key player in the negativefeedback regulation of NF-�B signaling in response tomultiple stimuli. NF-�B has an important role in immu-nity, and inappropriate NF-�B activity has been linkedto many autoimmune and inflammatory diseases (36).Our data revealed a trend toward decreased expressionof TNFAIP3 in EPC-derived cells from patients with SSccompared with controls. Consistent with this result,reduced TNFAIP3 expression was observed in the skinof patients with SSc. Taken together, these data suggestan impaired regulation of NF-�B, thereby promoting aninflammatory/immune response.

Our study has some limitations that deserveconsideration. The sample size can be regarded as small,and limitations inherent to microarray technology, pro-cessing, and analysis should also been taken into account(37,38). We focused only on the gene expression profileof EPC-derived endothelial cells. One perspective wouldbe to assess the exonic expression profiling of these cells,because we used Affymetrix Exon Arrays, which identify,in addition to gene expression, alternative splicing. Thesmall number of patients with dcSSc and patients withlcSSc made the comparison of their gene expressionprofiles less firm, especially because they were notmatched for disease duration. Another limitation is theabsence of an estimation of the family-wise error rateand the false-discovery rate, because the application ofthese correction methods precluded the identification ofpotentially relevant genes. Thus, we performed uncor-rected analyses to include the highest number of genesof interest but applied a stringent step of validation withquantitative RT-PCR and Western blot analyses.

Our data reveal important gene expressionchanges in late-outgrowth EPC-derived endothelial cellsfrom patients with SSc compared with control subjects,which might lead to a proadhesive, proinflammatory,and activated profile of these cells, contributing to theprocesses underlying SSc vasculopathy. In addition,hypoxia, one of the main characteristics of SSc, modu-lates the gene expression profile of EPC-derived endo-thelial cells and may contribute to their activated phe-notype in SSc. Moreover, we observed differential

expression of several new targets in SSc lesional skintissue, such as TNF family members and HOX-A9,which may contribute directly to the pathogenesis of thissevere incurable disease. These contributors warrantmore in-depth functional exploration, which could leadto potential new therapeutic targets and diagnosticmarkers.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Drs. Avouac and Allanore had fullaccess to all of the data in the study and take responsibility for theintegrity of the data and the accuracy of the data analysis.Study conception and design. Avouac, Allanore.Acquisition of data. Avouac, Cagnard, Schoindre, Ruiz, Couraud,Uzan, Boileau, Chiocchia, Allanore.Analysis and interpretation of data. Avouac, Cagnard, Distler,Chiocchia, Allanore.

ROLE OF THE STUDY SPONSOR

The funding agency had no role in the study design, datacollection, data analysis, and writing of the manuscript. The fundingagency approved the content of the submitted manuscript, but publi-cation was not contingent on approval by the funding agency.

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