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Molecular Medicine

, Volume 10, Issue 1–6, pp 19–27 | Cite as

Gene Expression Profile in Interleukin-4-Stimulated Human Vascular Endothelial Cells

  • Yong Woo Lee
  • Sung Yong Eum
  • Kuey Chu Chen
  • Bernhard Hennig
  • Michal Toborek
Open Access
Articles

Abstract

Interleukin-4 (IL-4)-mediated pro-oxidative and pro-inflammatory vascular environments have been implicated in the pathogenesis of atherosclerosis. The cellular and molecular regulatory mechanisms underlying this process, however, are not fully understood. In the present study, we employed GeneChip microarray analysis to investigate global gene expression patterns in human vascular endothelial cells after treatment with IL-4. Our results showed that mRNA levels of a total of 106 genes were significantly up-regulated and 41 genes significantly down-regulated with more than a 2-fold change. The majority of these genes are critically involved in the regulation of inflammatory responses, apoptosis, signal transduction, transcription factors, and metabolism; functions of the remaining genes are unknown. The changes in gene expression of selected genes related to inflammatory reactions, such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6), were verified by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. IL-4 treatment also significantly increased the adherence of inflammatory cells to endothelial cell monolayers in a dose-dependent manner. These results may help determine the molecular mechanisms of action of IL-4 in human vascular endothelium. In addition, a better understanding of IL-4-induced vascular injury at the level of gene expression could lead to the identification of new therapeutic strategies for atherosclerosis.

Introduction

Inflammatory responses elicited by a variety of stimuli in the vascular endothelium have been implicated in the development of cardiovascular disease. It is now widely believed that atherosclerosis is an inflammatory disease of the vessel wall, and inflammatory reactions in endothelial cells are primarily regulated through the production of inflammatory mediators and their close interactions (1). In fact, enhanced expressions of adhesion molecules, chemokines, and pro-inflammatory cytokines in vascular endothelial cells facilitate recruiting and adhering of inflammatory cells, such as lymphocytes and monocytes/macrophages, into the vessel wall, and thus stimulate transendothelial migration, which can be considered an early atherogenic process (2, 3, 4, 5). These studies strongly support the idea that an inflammatory environment in the vascular endothelium is critical for the initiation and development of atherosclerosis.

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T-helper 2 lymphocytes, eosinophils, and mast cells (6,7). IL-4 is present at high levels in tissues of patients with chronic inflammatory diseases, where it may play a critical role in the disease progression. Indeed, elevated levels of IL-4 were detected in atherosclerotic lesions (8). Additionally, a growing body of evidence indicates that IL-4 may play a role in atherogenesis through induction of inflammatory responses, such as up-regulation of vascular cell adhesion molecule-1 (VCAM-1) (9,10) and monocyte chemoattractant protein-1 (MCP-1) (11,12). IL-4 may also be considered as a pro-oxidative cytokine, which can increase the oxidative potential of target cells (10,13,14).

It has been proposed that the IL-4-mediated overexpression of inflammatory mediators is regulated at the transcriptional level through activation of a variety of redox-responsive transcription factors. For example, we have shown that IL-4-induced oxidative stress up-regulates the expression of VCAM-1 and MCP-1 genes via activation of Sp-1 and signal transducers and activators of transcription, respectively (10,12). Although previous studies have established the potential role of IL-4 in the development of cardiovascular disease, the cellular and molecular regulatory mechanisms underlying this process are not fully understood. In the present study, GeneChip microarray analysis was conducted to investigate the global gene expression changes of IL-4-treated human vascular endothelial cells using the Affymetrix GeneChip® Human Genome U133A Arrays. In addition, we also employed the quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) to confirm changes in the levels of expression of selective genes of interest. We found that IL-4 significantly regulates the expression of genes known to be involved in inflammation, apoptosis, signal transduction, and transcription factors.

Materials and Methods

Cell Cultures

Human umbilical vein endothelial cells (HUVEC) were isolated as described previously (15). HUVEC were cultured in enriched M199 medium supplemented with 20% fetal calf serum, 1% each of penicillin/streptomycin, glutamine, and antibiotic-antimycotic, heparin (300 µg/mL; Gibco BRL, Grand Island, NY, USA), HEPES (6 mg/mL; Sigma Chemical, St. Louis, MO, USA), and endothelial cell growth supplement (40 µg/mL; Collaborative Research, Bedford, MA, USA) in 5% CO2 at 37 °C. Cells were determined to be endothelial by their cobblestone morphology and uptake of fluorescent-labeled acetylated LDL (1,1’-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate; Molecular Probes Inc., Eugene, OR, USA). HUVEC from passage 2 were used in all experiments. The human monocytic leukemia cells (THP-1) were purchased from American Type Culture Collection (Manassas, VA, USA) and used to study cell adhesion assay. THP-1 cells were cultured in suspension in RPMI 1640 medium supplemented with 10% fetal calf serum, 25 mM glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 50 µM 2-mercaptoethanol, and 1% each of penicillin/ streptomycin in 5% CO2 at 37 °C.

GeneChip Microarray Analysis

Microarray gene expression analysis was performed using the Affymetrix GeneChip System with Human Genome U133A Arrays (Affymetrix Inc, Santa Clara, CA, USA).

RNA isolation and GeneChip microarray processing. HUVEC were either untreated or treated with 10 ng/mL of IL-4 for 4 h. Total RNA was isolated and purified using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocols. The labeling of RNA samples, human GeneChip (HG-U133A) hybridization, and array scanning were carried out as described earlier (16,17) and according to the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, an average yield of 40 µg of biotin-labeled cRNA target was obtained from 5 µg of total RNA from each sample, of which 20 µg of cRNA was applied to 1 gene chip. The hybridization was run overnight in a rotating oven (Affymetrix GeneChip Hybridization Oven 640) at 45 °C. The chips were then washed and stained on a fluidics station (Affymetrix GeneChip Fluidics Station 400), and scanned at a resolution of 3 µm in a confocal scanner (Affymetrix GeneArray Scanner).

Microarray data analysis. The gene expression levels of samples were analyzed using the Affymetrix Microarray Suite software according to the manufacturer’s recommendation. All data presented in Tables 1 and 2 show the mean fold change in gene expression from 3 independent experiments in IL-4-treated HUVEC compared with untreated control cell cultures. All genes presented were significantly changed (P < 0.05) and the mean fold minimum change chosen for presentation was 2.0.
Table 1

Up-regulation of specific gene expression in human vascular endothelial cells treated with interleukin-4

Gene symbol

NCBI accession nr

Fold change

P value

Description

Adhesion molecules

    

VCAM1

NM_001078

21.7

<0.001

Vascular cell adhesion molecule 1

CSPG2

D32039

5.4

0.006

Chondroitin sulfate proteoglycan 2

SELE

NM_000450

4.3

<0.001

E-selectin

AIM1

U83115

4.1

<0.001

Absent in melanoma 1

AGC1

X17406

3.2

0.002

Aggrecan 1

PCDH7

NM_002589

2.7

<0.001

BH-protocadherin

FCN3

NM_003665

2.2

0.023

Ficolin (collage/fibrinogen domain) 3

FAT

NM_005245

2.0

0.007

FAT tumor suppressor homolog 1

Chemokines and cytokines

    

CCL2

S69738

8.2

<0.001

Monocyte chemoattractant protein-1

IL6

NM_000600

2.0

0.003

Interleukin-6

Apoptosis

    

PAWR

NM_002583

2.6

0.001

PRKC, apoptosis, WT1, regulator

CASP3

NM_004346

2.5

0.008

Caspase 3

CASP2

AF314174

2.0

0.028

Caspase 2

Signal transduction

    

PMCH

NM_002674

331.2

<0.001

Promelanin-concentrating hormone

PIK3CG

AF327656

9.9

0.013

Phosphoinositide-3-kinase, catalytic, γ

SOCS1

AB005043

5.8

<0.001

Suppressor of cytokine signaling 1

LIFR

NM_002310

5.7

<0.001

Leukemia inhibitory factor receptor

BMP4

D30751

3.4

<0.001

Bone morphogenetic protein 4

CSF2RB

AV756141

3.2

<0.001

Colony stimulating factor 2 receptor β

KIAA0551

AF172268

3.2

<0.001

Traf2 and NCK interacting kinase

RGS2

NM_002923

3.1

<0.001

Regulator of G-protein signaling 2

INHBA

M13436

3.0

<0.001

Inhibin βA

MET

BG170541

2.7

0.003

Hepatocyte growth factor receptor

RICS

NM_014715

2.3

0.045

Rho GTPase-activating protein

H11

AF133207

2.2

0.002

Protein kinase H11

BMP2

NM_001200

2.2

<0.001

Bone morphogenetic protein 2

GUCY1B3

W93728

2.1

0.010

Guanylate cyclase 1, soluble, β3

EXT1

NM_000127

2.0

<0.001

Exostoses (multiple) 1

ARL7

NM_005737

2.0

0.005

ADP-ribosylation factor-like 7

Transcription factors

    

CREM

D14826

8.5

0.004

cAMP responsive element modulator

PKNOX2

AK023792

6.2

0.001

PBX/knotted 1 homeobox 2

MAD

NM_002357

5.4

<0.001

MAX dimerization protein 1

FOXC1

AU145890

4.6

0.011

Forkhead box C1

CITED2

NM_006079

4.2

<0.001

Cbp/p300-interacting transactivator

IRLB

BE268538

4.1

<0.001

c-Myc promoter-binding protein

CEBPB

AL564683

2.9

<0.001

CCAAT/enhancer binding protein β

POU4F1

NM_006237

2.6

0.005

POU domain, class 4, transcription factor 1

GATA6

D87811

2.3

0.018

GATA binding protein 6

KIAA0146

NM_005195

2.3

<0.001

CCAAT enhancer binding protein (CEBP)

SSRP2

NM_012446

2.2

0.003

Single-stranded DNA binding protein 2

ELL2

NM_012081

2.1

0.022

ELL-related RNA polymerase II, elongation factor

KLF4

BF514079

2.1

0.011

Kruppel-like factor 4

TRAP95

NM_005481

2.0

0.038

Thyroid hormone receptor-associated protein, 95-kD subunit

CART1

NM_006982

2.0

0.024

Cartilage paired-class homeoprotein 1

TOX

AI961231

2.0

0.009

Thymus high mobility group box protein

Others

    

MCHP

S64288

204.2

<0.001

Melanin-concentrating hormone precursor

HS3ST1

BF000296

10.7

0.002

Heparan sulfate 3-O-sulfotransferase 1

CDC45L

NM_003504

7.4

0.003

CDC45 cell division cycle 45-like

SIAT8A

L32867

6.5

<0.001

Sialyltransferase 8A

TMOD1

NM_003275

5.9

0.004

Tropomodulin 1

MTHFR

AJ249275

5.7

0.009

5, 10-Methylenetetrahydrofolate reductase

ENPP1

NM_006208

5.2

0.004

Ectonucleotide pyrophosphatase/phosphodiesterase 1

LOX

NM_002317

5.1

<0.001

Lysyl oxidase

AMIGO4

AC004010

4.2

<0.001

Amphoterin induced gene 2

SULF1

AW043713

4.6

<0.001

Sulfatase 1

GJA5

NM_005266

4.3

<0.001

Gap junction protein (Connexin 40)

ARK5

NM_014840

3.9

<0.001

KIAA0537 gene product

FKBP5

NM_004117

3.8

0.002

FK506 binding protein 5

LRRTM2

NM_015564

3.8

0.017

Leucine-rich repeat transmembrane neuronal 2 protein

COVA1

NM_006375

3.6

0.014

Cytosolic ovarian carcinoma antigen 1

DACT1

NM_016651

3.6

<0.001

Dapper homolog 1, antagonist of β-catenin

DMD

NM_004010

3.6

<0.001

Dystrophin

LRRN3

AI221950

3.5

0.006

Leucine rich repeat neuronal 3

PTX3

NM_002852

3.5

<0.001

Pentaxin-related gene

CLCN4

AA071195

3.5

0.006

Chloride channel 4

SLC38A1

NM_030674

3.5

<0.001

Amino acid transporter system A1

FLJ11743

NM_024527

3.5

0.011

Hypothetical protein FLJ11743

OSPBL11

NM_022776

3.4

<0.001

Oxysterol binding protein-like 11

CH25H

NM_003956

3.2

0.026

Cholesterol 25-hydroxylase

FBN1

AI264196

2.8

0.003

Fibrillin 1 (Marfan syndrome)

ELAVL2

NM_004432

2.7

<0.001

ELAV (embryonic lethal, abnormal vision)-like 2

CPT2

M58581

2.7

0.020

Carnitine palmitoyltransferase II

NNMT

NM_006169

2.5

<0.001

Nicotinamide N-methyltransferase

UP

NM_003364

2.5

<0.001

Uridine phosphorylase

KCNK3

NM_002246

2.5

0.022

Potassium channel, subfamily K, member 3

DAAM1

AK021890

2.5

<0.001

Dishevelled associated activator of morphogenesis 1

SLC22A4

NM_003059

2.4

<0.001

Solute carrier family 22, member 4

PSCD1

NM_004762

2.4

<0.001

Cytohesin 1

JAG1

NM_000214

2.4

0.024

Jagged 1 (Alagille syndrome)

EGLN3

NM_022073

2.4

0.007

Egl 9 homolog 3

TPK1

NM_022445

2.3

0.005

Thiamin pyrophosphokinase 1

LOC169611

AL050002

2.3

0.004

Hypothetical protein LOC169611

PVRL3

AA129716

2.3

0.004

Poliovirus receptor-related 3

DOK5

AL050069

2.2

0.002

Docking protein 5

MRF2

BG285011

2.2

<0.001

Modulator recognition factor 2

DNAJC3

NM_006260

2.2

0.034

DnaJ (Hsp40) homolog, subfamily C, 3

CPR8

AK022459

2.2

0.005

Cell cycle progression 8 protein

SIAT1

AV695711

2.1

0.002

Sialyltransferase 1

RDX

NM_002906

2.1

0.024

Radixin

CYP1B1

NM_000104

2.0

0.010

Cytochrome P450 1B1

ALDH1A2

NM_003888

2.0

0.002

Aldehyde dehydrogenase1A2

GCNT1

NM_001490

2.0

<0.001

β-1,6-N-acetylglucosaminyltransferase

Unknown

    

DKK2

NM_014421

26.5

<0.001

Dickkopf homolog 2

T12479

AW029169

8.5

<0.001

Hypothetical protein DKFZp 564N1362.1

FLJ10713

NM_018189

5.0

<0.001

Hypothetical protein FLJ10713

KIAA0977

NM_014900

3.8

<0.001

KIAA0977 protein

LOC51334

NM_016644

2.8

0.010

Mesenchymal stem cell protein DSC54

CGI-115

NM_016052

2.6

<0.001

CGI-115 protein

C13orf7

NM_024546

2.4

<0.001

Chromosome 13 open reading frame 7

FLJ20378

AI336206

2.2

<0.001

Hypothetical protein FLJ20378

SCA1

NM_000332

2.2

0.012

Spinocerebellar ataxia 1

FLJ90005

W27419

2.1

<0.001

Hypothetical protein FLJ90005

24739

AF070571

2.1

0.011

Homo sapiens clone 24739

FJX1

NM_014344

2.1

0.007

Four jointed box 1

FLJ10901

NM_018265

2.0

0.020

Hypothetical protein FLJ10901

HRASLS

NM_020386

2.0

0.002

HRAS-like suppressor

Housekeeping genes

    

ACTB

Hs.426930

1.0

0.694

β-Actin

GAPDH

M33197

1.0

0.680

Glyceraldehyde-3-phosphate dehydrogenase

Table 2

Down-regulation of specific gene expression in human vascular endothelial cells treated with interleukin-4

Gene symbol

NCBI accession nr

Fold change

P value

Description

Adhesion molecules

    

ITGA2

NM_002203

−3.6

<0.001

Integrin α2

Cytokines, chemokines, and receptors

    

IL8

NM_000584

−5.6

<0.001

Interleukin-8

IL7R

NM_002185

−3.4

0.005

Interleukin-7 receptor

CXCL2

M57731

−2.2

0.001

Chemokine (C-X-C motif) ligand 2

Growth factors and receptors

    

GAS1

NM_002048

−14.0

0.007

Growth arrest-specific 1

NDRG4

AV724216

−2.1

0.011

NDRG family member 4

EGFR

AW157070

−2.1

0.006

Epidermal growth factor receptor

PDGFB

NM_002608

−2.0

0.029

Platelet-derived growth factor β polypeptide

Signal transduction

    

KIT

NM_000222

−9.0

<0.001

v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog

ITPKB

NM_002221

−2.6

0.001

Inositol 1,4,5-triphophate 3-kinase B

RAB11B

AL575337

−2.2

0.016

RAB11B, member RAS oncogene family

TNS

AL046979

−2.0

0.036

Tensin

Transcription factors

    

KLF15

NM_014079

−2.7

0.004

Kruppel-like factor 15

SNAPC4

AK023513

−2.2

0.002

Small nuclear RNA activating complex, polypeptide 4, 190 kDa

NPAS2

AW000928

−2.1

<0.001

Neuronal PAS domain protein 2

HOXB6

NM_018952

−2.0

0.011

Homeo box B6

Others

    

GJA4

NM_002060

−4.8

0.006

Gap junction protein (connexin 37)

HIP14

AF161412

−2.9

0.019

Huntingtin interacting protein 14

KCNN2

NM_021614

−2.8

<0.001

Potassium intermediate/small conductance calcium-activated channel N2

AMN

NM_030943

−2.6

0.029

Amnionless homolog

NFNG

AI760053

−2.6

0.023

Manic fringe homolog

CLTB

X81637

−2.5

0.032

Clathrin light chain b gene

CHST2

NM_004267

−2.2

<0.001

Carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2

FLJ12800

NM_022903

−2.2

0.030

Hypothetical protein FLJ12800

CLDN18

BE551219

−2.2

0.046

Claudin 18

RASGRP3

NM_015376

−2.1

0.003

RAS guanine releasing protein 3

CYP2A6

NM_000762

−2.1

<0.001

Cytochrome P450 2A6

TRPV5

NM_019841

−2.1

0.008

Trasient receptor potential cation channel, subfamily V, member 5

CAV3

NM_001234

−2.0

0.021

Caveolin 3

BPAG1

BG253119

−2.0

0.031

Bullous pemphigoid antigen 1

WIZ

AL390184

−2.0

0.009

Widely interspaced zinc finger motifs

Unknown

    

CHI3L1

AJ251847

−4.3

0.005

CHI3L1 gene for cartilage glycoprotein-39

R29124_1

BF110434

−3.3

0.004

Hypothetical protein R29124_1

FLJ11983

AK022045

−2.9

0.004

FLJ11983 fis, clone HEMBA1001337

PRO1598

NM_018503

−2.7

0.022

Hypothetical protein PRO1598

FLJ10002

AK000864

−2.6

0.007

FLJ10002 fis, clone HEMBA1000046

FLJ23497

NM_025089

−2.6

0.044

Hypothetical protein FLJ23497

KIAA0795

NM_025010

−2.5

0.013

KIAA0795 protein

HL14

M14087

−2.4

0.049

HL14 gene encoding β-galactoside-binding lectin

FLJ20378

AI734156

−2.2

0.033

Hypothetical protein FLJ20378

LOC92346

AL035295

− 2.0

0.040

PAC 106H8, similar to Dynamin

Real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Quantitative real-time RT-PCR, also known as fluorescence-based kinetic RT-PCR, was employed to confirm specific gene expression changes detected by the GeneChip analysis. The fluorogenic 5′ nuclease assay technology using TaqMan® probes was used to ensure specificity and sensitivity. Total RNA was isolated and purified using RNeasy Mini Kit (Qiagen) according to the protocol of the manufacturer. Then, 1 µg of total RNA was reverse-transcribed at 25 °C for 15 min, 42 °C for 45 min, and 99 °C for 5 min in 20 µL of 5 mM MgCl2, 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1 mM dNTP, 1 unit/µL of recombinant RNasin ribonuclease inhibitor, 15 units/µg of AMV reverse transcriptase, and 0.5 µg of random hexamers. For quantitative PCR, amplifications of individual genes were performed on ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using TaqMan® Universal PCR Master Mix, gene-specific TaqMan PCR probes and primers, and a standard thermal cycler protocol (50 °C for 2 min before the 1st cycle, 95 °C for 15 s, and 60 °C for 1 min, repeated 45 times). For specific probes and primers of PCR amplifications, Assay-on-DemandTM Products for human VCAM-1 and E-selectin, and TaqMan Pre-Developed Assay Reagents for human MCP-1, IL-6, and β-actin, were obtained from Applied Biosystems. The threshold cycle (CT), which indicates the fractional cycle number at which the amount of amplified target gene reaches a fixed threshold, from each well was determined using ABI Prism 7000 SDS software. Relative quantification, which represents the change in gene expression from real-time quantitative PCR experiments between IL-4-treated group and untreated control group, was calculated by the comparative CT method as described earlier (18,19). The data were analyzed using equation 2−∆∆CT, where ∆∆CT = [CT of target gene — CT of housekeeping gene]treated group — [CT of target gene — CT of housekeeping gene]untreated control group. For the treated samples evaluation of 2−∆∆CT represents the fold change in gene expression, normalized to a housekeeping gene (β-actin) and relative to the untreated control.

Enzyme-linked Immunosorbent Assay (ELISA)

Cell surface expression levels of adhesion molecules such as VCAM-1 and E-selectin were quantified by ELISA Development kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s procedure, with modifications. Briefly, HUVEC monolayers were incubated with either anti-human VACM-1 or E-selectin monoclonal antibody (2.5 µg/mL) for 1 h at 37 °C. The cells were then incubated with biotinylated goat antimouse IgG antibody (1:1,000 dilution) for 1 hat37 °C. After washing the wells thoroughly, the working dilution of Streptavidin-HRP was added to each well and incubated for 20 min at room temperature. The cells were incubated with HRP Substrate Solution for 20 min at room temperature with subsequent addition of Stop Solution. After color development, absorbance from each well was measured by a microtiter plate reader at 450 nm to 570 nm.

The protein levels of human MCP-1 and IL-6 in cell culture supernatants were determined using Human MCP-1 Immunoassay and Human IL-6 Immunoassay kits (R&D Systems) according to the protocol of the manufacturer, respectively. This assay employs the quantitative sandwich enzyme immunoassay technique using a murine monoclonal antibody against human MCP-1 or IL-6, and a polyclonal secondary antibody conjugated with horseradish peroxidase. The minimum detectable concentration of MCP-1 and IL-6 was less than 5.0 and 0.70 pg/mL, respectively.

Cell Adhesion Assay

Adhesion studies were performed with the human monocytic leukemia cell line, THP-1, as previously described (20) with modifications (21). Briefly, HUVEC were grown to confluence on 24-well plates and exposed to IL-4 for 8 and 24 h. Prior to the cell-cell adhesion assay, the HUVEC monolayers were washed twice with Hank’s Balanced Salt Solution (HBSS) and then washed with M199 medium containing 10% fetal bovine serum. Calcein acetoxymethyl ester (calcein AM; Calbiochem, La Jolla, CA, USA) was employed to label THP-1 cells. The fluorescence labeling of THP-1 cells was achieved by incubating cells (2.5 × 105 cells/mL) with 5 µg/mL of calcein AM. After loading of calcein AM for 20 min at 37 °C, the cells were washed 3 times with HBSS, and then washed with M199 medium containing 10% fetal bovine serum. The calcein AM-labeled THP-1 cells were added onto the HUVEC monolayers and incubated for 20 min at 37 °C. The non-adherent THP-1 cells were removed from monolayers by washing each well 3 times with HBSS. The fluorescence intensity was measured by a fluorescence plate reader using excitation of 490 nm and emission of 517 nm.

Statistical Analysis

Routine statistical analysis of data was completed using Sigma-Stat 2.03 (SPSS, Chicago, IL, USA). Statistical probability of P < 0.05 was considered significant.

Results

IL-4 Up-regulates Adhesion of Leukocytes to Human Vascular Endothelial Cell Monolayers

The adherence of human acute monocytic leukemia cells, THP-1, to HUVEC monolayers was determined to verify the functional integrity of inflammatory mediators up-regulated by human vascular endothelial cells after stimulation with IL-4. Following an 8-to 24-h incubation with IL-4 doses ranging from 0.1 to 10 ng/mL, endothelial cell function was significantly and dose-dependently altered as assessed by changes in THP-1 adherence to the HUVEC monolayer (Figure 1). Hence, endothelial cells were exposed to 10 ng/mL of IL-4 for 4 h and changes in gene expression were assessed using microarray analysis.
Figure 1

IL-4 up-regulates the adhesion of leukocytes to human vascular endothelial cell monolayers. HUVEC were either untreated or treated with the indicated concentrations of IL-4 (0.1, 1.0, and 10 ng/mL) for up to 24 h. The adherence of calcein AM-labeled THP-1 cells was measured by fluorescent microplate reader using excitation of 490 nm and emission of 517 nm. Data are means ± SD of 4 determinations. *Statistically significant compared with the control group (P < 0.05).

Identification of Global Gene Expression Changes in IL-4-Treated Human Vascular Endothelial Cells

The gene expression profile of human vascular endothelial cells treated with IL-4 was assessed using microarray technology with the Affymetrix GeneChip Human Genome U133A Arrays, which contain more than 22000 human genes. As shown in Table 1, 106 genes were significantly up-regulated at the mRNA level with more than a 2-fold change in HUVEC after treatment with IL-4 for 4 h. Classification by function revealed that IL-4 treatment up-regulated genes mainly responsible for inflammatory reactions, apoptosis, signal transduction, and transcription factors. Among these, mRNA levels of the inflammatory mediators, such as adhesion molecules (VCAM-1 and E-selectin), chemokine (MCP-1), and pro-inflammatory cytokine (IL-6), were markedly and significantly induced, suggesting that IL-4 can play a crucial role in the pro-inflammatory pathways in human vascular endothelium. The expression of 2 housekeeping genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, was not affected with IL-4 treatment. In addition, exposure of HUVEC to IL-4 resulted in a significant down-regulation of 41 genes by at least 2-fold factor, as compared with untreated control cell cultures (Table 2).

Verification of Microarray Analysis Using Real-Time RT-PCR and ELISA

To validate the changes in gene expression of IL-4-treated human vascular endothelial cells observed in microarray analysis, we performed quantitative real-time RT-PCR for several target genes that were up-regulated in HUVEC treated with IL-4. In the present study, we selected 4 inflammatory genes encoding VCAM-1, E-selectin, MCP-1, and IL-6. Real-time RT-PCR showed that increasing concentrations of IL-4 dramatically induced mRNA expression of adhesion molecules, such as VCAM-1 and E-selectin (Figures 2A and 2B). A significant and dose-dependent induction of chemokine MCP-1 gene was also observed in HUVEC treated with IL-4 (see Figure 2C). Additionally, IL-4 treatment markedly up-regulates gene expression of pro-inflammatory cytokine IL-6 (see Figure 2D). These results confirm that up-regulation of selected genes identified by microarray analysis correlates with mRNA expression measured by real-time RT-PCR. In parallel with gene expression analyses, a series of ELISA was conducted to determine whether IL-4-induced increases in mRNA levels could translate to elevated protein expression. Consistent with the data on gene expression, treatment with IL-4 resulted in a significant and dose-dependent up-regulation of protein expression of VCAM-1, E-selectin, MCP-1, and IL-6 (Figures 3A to 3D).
Figure 2

IL-4 up-regulates the mRNA expression of inflammatory mediators in human vascular endothelial cells. HUVEC were either untreated or treated with the indicated concentrations of IL-4 (0.1, 1.0, and 10 ng/mL) for 4 h. The mRNA levels of VCAM-1 (A), E-selectin (B), MCP-1 (C), and IL-6 (D) were determined by real-time RT-PCR as described in Materials and Methods. Data are means ± SE of 4 determinations. *Statistically significant compared with the control group (P < 0.05).

Figure 3

IL-4 up-regulates the protein expression of inflammatory mediators in human vascular endothelial cells. HUVEC were either untreated or treated with the indicated concentrations of IL-4 (0.1, 1.0, and 10 ng/mL) for 12 h (VCAM-1), 6 h (E-selectin), or 16 h (MCP-1 and IL-6). The protein levels of VCAM-1 (A), E-selectin (B), MCP-1 (C), and IL-6 (D) were measured by ELISA as described in Materials and Methods. Data are means ± SD of 4 determinations. *Statistically significant compared with the control group (P < 0.05).

Discussion

Microarray analysis is one of the most advanced and emerging molecular biological technologies, and it has been widely adopted for analyzing the global gene expression profiles in vivo and in vitro (22,23). Recent studies have demonstrated the potential of this technology for investigating molecular pathophysiological mechanisms involved in a variety of human diseases. In fact, microarray technology has been used as a novel experimental approach to analyze alterations in gene expression in cancer (24), atherosclerosis (25), stroke (26), Alzheimer’s disease (27), HIV infection (28), schizophrenia (29), and muscular dystrophy (30).

In the present study, we performed microarray analysis using the Affymetrix GeneChip Human Genome U133A Arrays to further understand transcriptional regulatory mechanisms of action of IL-4 in human vascular endothelium. Our results revealed that mRNA levels of a total of 106 genes were significantly up-regulated and 41 genes significantly down-regulated with more than a 2-fold change in HUVEC treated with IL-4 compared with the control cell cultures (see Tables 1 and 2). Interestingly, many of IL-4-up-regulated genes are involved in inflammatory reactions, which are critical to initiate and promote early stage of atherogenesis. Previous studies from our group and others have demonstrated that IL-4-induced oxidative stress can produce a proinflammatory vascular environment through up-regulation of inflammatory genes, such as adhesion molecules, chemokines, and cytokines (9, 10, 11, 12,31,32). The present data, showing significant up-regulation of VCAM-1, E-selectin, MCP-1, and IL-6, strongly support that IL-4 is a key mediator to induce pro-oxidative and pro-inflammatory pathways in human vascular endothelium.

To verify the alterations in gene expression observed in microarray analysis, as well as to further explore the potential role of IL-4 in inflammatory pathways in human vascular endothelium, the present study focused on a set of genes related to inflammatory reactions such as VCAM-1, E-selectin, MCP-1, and IL-6. VCAM-1 is expressed primarily on endothelial cells and mediates cell-cell interactions via binding to its integrin counter receptor, very late antigen-4, which may be involved in the recruitment of mononuclear leukocytes to the vascular lesions in early atherosclerosis (33). We and others have shown that IL-4 up-regulates VCAM-1 expression in vascular endothelial cells through antioxidant-sensitive mechanisms (10,31,32). In agreement with previous studies, a marked and significant increase in mRNA and protein expression of VCAM-1 was observed in IL-4-treated HUVEC by real-time RT-PCR and ELISA, respectively (see Figures 2A and 3A).

Another adhesion molecule studied in the present study was E-selectin. E-selectin is present exclusively on the surface of endothelial cells and plays a key role in mediating early leukocyte-endothelial interactions such as initial attachment and rolling during an inflammatory response. It is well documented that E-selectin is up-regulated at the transcriptional level following exposure to a series of pro-inflammatory mediators, such as IL-1β, TNF-α, and lipopolysaccharide (34). In contrast, it has been proposed that treatment of endothelial cells with IL-4 suppresses IL-1β- or TNF-α-stimulated E-selectin gene transcription (35,36). Direct effects of IL-4 on E-selectin expression in human vascular endothelial cells, however, remain unclear. In the present study, we provide new evidence to indicate that IL-4 could directly upregulate mRNA and protein expression of E-selectin in HUVEC (see Figures 2B and 3B). These results suggest that E-selectin may play an important role in IL-4-mediated inflammatory pathways in vascular endothelium.

Among a variety of chemokines and inflammatory cytokines, MCP-1 and IL-6 are of critical significance in the early stages of atherosclerosis. MCP-1 is secreted by a variety of cell types, including vascular endothelial cells, and promotes the recruitment of inflammatory cells and their migration throughout the vascular endothelium that are thought to be critical early pathological events in atherogenesis (37,38). Consistent with previous experiments (11,12), the present study showed that IL-4 treatment resulted in up-regulation of mRNA and protein expression of MCP-1 in human vascular endothelial cells (see Figures 2C and 3C).

IL-6, a multifunctional pro-inflammatory cytokine, plays a major role in inflammatory responses in vascular endothelium and has also been implicated in the pathogenesis of atherosclerosis (1,39). Although recent evidence indicates that IL-4 synergistically amplifies the TNF-α-, IL-1β-, or LPS-induced production of IL-6 protein in HUVEC (40), the molecular basis for the induction of this cytokine by IL-4 has not been elucidated. Therefore, our results, showing that IL-4 significantly induced the expression of IL-6 mRNA and increased IL-6 production in HUVEC (Figures 2D and 3D), appear to be the first to document the stimulatory effect of IL-4 on IL-6 gene expression in human vascular endothelial cells.

In conclusion, the present study provides the first quantitative large-scale gene expression analysis of IL-4-stimulated human vascular endothelial cells. We identified 147 differentially regulated genes that are responsible for the regulation of inflammatory responses, apoptosis, signal transduction, transcription factors, metabolism, and several unknown functions. Because IL-4 is involved in the early stages of atherogenesis, these results could contribute to a deeper understanding of fundamental insights of pathophysiological mechanisms involved in atherosclerosis at the level of gene expression and provide a foundation for development of therapeutic strategies for vascular diseases.

Notes

Acknowledgments

This work was supported by grants from the American Heart Association, Ohio Valley Affiliate, NIEHS/NIH (ES07380), and the University of Kentucky Microarray Facility Program.

References

  1. 1.
    Ross R. (1999) Atherosclerosis is an inflammatory disease. Am. Heart. J. 138:S419–20.CrossRefGoogle Scholar
  2. 2.
    Davies MJ et al. (1993) The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J. Pathol. 171:223–9.CrossRefGoogle Scholar
  3. 3.
    Cotran RS, Mayadas-Norton T. (1998) Endothelial adhesion molecules in health and disease. Pathol. Biol. 46:164–70.PubMedGoogle Scholar
  4. 4.
    Reape TJ, Groot PH. (1999) Chemokines and atherosclerosis. Atherosclerosis 147:213–25.CrossRefGoogle Scholar
  5. 5.
    Rus HG, Niculescu F, Vlaicu R. (1991) Tumor necrosis factor-alpha in human arterial wall with atherosclerosis. Atherosclerosis 89:247–54.CrossRefGoogle Scholar
  6. 6.
    Paul WE. (1991) Interleukin-4: A prototypic immunoregulatory lymphokine. Blood 77:1859–70.PubMedGoogle Scholar
  7. 7.
    Rocken M, Racke M, Shevach EM. (1991) IL-4-induced immune deviation as antigen-specific therapy for inflammatory autoimmune disease. Immunol. Today 17:225–31.CrossRefGoogle Scholar
  8. 8.
    Sasaguri T et al. (1998) A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells. Atherosclerosis 138:247–53.CrossRefGoogle Scholar
  9. 9.
    Galea P, Chartier A, Lebranchu Y. (1991) Increased lymphocyte adhesion to allogeneic endothelial cells by interleukin-4 (IL-4). Transplant Proc. 23:243–44.PubMedGoogle Scholar
  10. 10.
    Lee YW, Kühn H, Hennig B, Neish AS, Toborek M. (2001) IL-4-induced oxidative stress upregulates VCAM-1 gene expression in human endothelial cells. J. Mol. Cell. Cardiol. 33:83–94.CrossRefGoogle Scholar
  11. 11.
    Rollins BJ, Pober JS. (1991) Interleukin-4 induces the synthesis and secretion of MCP-1/JE by human endothelial cells. Am. J. Pathol. 138:1315–9.PubMedPubMedCentralGoogle Scholar
  12. 12.
    Lee YW, Hennig B, Toborek M. (2003) Redox-regulated mechanisms of IL-4-induced MCP-1 expression in human vascular endothelial cells. Am. J. Physiol. Heart Circ. Physiol. 284:H185–92.CrossRefGoogle Scholar
  13. 13.
    Brinckmann R et al. (1996) Regulation of 15-lipoxygenase expression in lung epithelial cells by interleukin-4. Biochem. J. 318:305–12.CrossRefGoogle Scholar
  14. 14.
    Lee YW et al. (2001) Interleukin 4 induces transcription of the 15-lipoxygenase I gene in human endothelial cells. J. Lipid Res. 42:783–91.PubMedGoogle Scholar
  15. 15.
    Toborek M, Lee YW, Kaiser S, Hennig B. (2002) Measurement of inflammatory properties of fatty acids in human endothelial cells. Methods Enzymol. 352:198–219.CrossRefGoogle Scholar
  16. 16.
    Blalock EM et al. (2003) Gene microarrays in hippocampal aging: Statistical profiling identifies novel processes correlated with cognitive impairment. J. Neurosci. 23:3807–19.CrossRefGoogle Scholar
  17. 17.
    Blalock EM et al. (2004) Incipient Alzheimer’s disease: Microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc. Natl. Acad. Sci. U.S.A. 101:2173–8.CrossRefGoogle Scholar
  18. 18.
    Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2∆∆CT method. Methods 25:402–8.CrossRefGoogle Scholar
  19. 19.
    Deng X, Li H, Tang YW. (2003) Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR. J. Virol. Methods. 107:141–6.CrossRefGoogle Scholar
  20. 20.
    Braut-Boucher F et al. (1995) A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes. J. Immunol. Methods. 178:41–51.CrossRefGoogle Scholar
  21. 21.
    Choi W et al. (2003) PCB 104-induced proinflammatory reactions in human vascular endothelial cells: Relationship to cancer metastasis and atherogenesis. Toxicol. Sci. 75:47–56.CrossRefGoogle Scholar
  22. 22.
    Watson A, Mazumder A, Stewart M, Balasubramanian S. (1998) Technology for microarray analysis of gene expression. Curr. Opin. Biotechnol. 9:609–14.CrossRefGoogle Scholar
  23. 23.
    Schulze A, Downward J. (2001) Navigating gene expression using microarrays—a technology review. Nature Cell Biol. 3:E190–5.CrossRefGoogle Scholar
  24. 24.
    Golub TR et al. (1999) Molecular classification of cancer: Class discovery and class prediction by gene expression monitoring. Science 286:531–7.CrossRefGoogle Scholar
  25. 25.
    Hiltunen MO et al. (2002) Changes in gene expression in atherosclerotic plaques analyzed using DNA array. Atherosclerosis 165:23–32.CrossRefGoogle Scholar
  26. 26.
    Bowler RP et al. (2002) A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke. Free Radic. Biol. Med. 33:947–61.CrossRefGoogle Scholar
  27. 27.
    Ginsberg SD, Hemby SE, Lee VM, Eberwine JH, Trojanowski JQ. (2000) Expression profile of transcripts in Alzheimer’s disease tangle-bearing CA1 neurons. Ann. Neurol. 48:77–87.CrossRefGoogle Scholar
  28. 28.
    Geiss GK et al. (2000) Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays. Virology. 266:8–16.CrossRefGoogle Scholar
  29. 29.
    Mirnics K, Middleton FA, Marquez A, Lewis DA, Levitt P. (2000) Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron. 28:53–67.CrossRefGoogle Scholar
  30. 30.
    Chen YW, Zhao P, Borup R, Hoffman EP. (2000) Expression profiling in the muscular dystrophies: Identification of novel aspects of molecular pathophysiology. J. Cell Biol. 151:1321–36.CrossRefGoogle Scholar
  31. 31.
    Masinovsky B, Urdal D, Gallatin WM. (1990) IL-4 acts synergistically with IL-1β to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1. J. Immunol. 145:2886–95.PubMedGoogle Scholar
  32. 32.
    Blease K, Seybold J, Adcock IM, Hellewell PG, Burke-Gaffney A. (1998) Interleukin-4 and lipopolysaccharide synergize to induce vascular cell adhesion molecule-1 expression in human lung microvascular endothelial cells. Am. J. Respir. Cell Mol. Biol. 18:620–30.CrossRefGoogle Scholar
  33. 33.
    Cybulsky M, Gimbrone M. (1991) Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 251:788–91.CrossRefGoogle Scholar
  34. 34.
    Whelan J. (1996) Selectin synthesis and inflammation. Trends Biochem. Sci. 21:65–9.CrossRefGoogle Scholar
  35. 35.
    Bennett BL, Cruz R, Lacson RG, Manning AM. (1997) Interleukin-4 suppression of tumor necrosis factor α-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-κB. J. Biol. Chem. 272:10212–9.CrossRefGoogle Scholar
  36. 36.
    Thornhill MH, Haskard DO. (1990) IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-γ. J. Immunol. 145:865–72.PubMedGoogle Scholar
  37. 37.
    Gu L, Tseng SC, Rollins BJ. (1999) Monocyte chemoattractant protein-1. Chem. Immunol. 72:7–29.CrossRefGoogle Scholar
  38. 38.
    Rollins BJ. (1997) Chemokines. Blood 90:909–28.PubMedGoogle Scholar
  39. 39.
    Libby P, Ridker PM, Maseri A. (2002) Inflammation and atherosclerosis. Circulation 105:1135–43.CrossRefGoogle Scholar
  40. 40.
    Chen CC, Manning AM. (1996) TGF-β1, IL-10 and IL-4 differentially modulate the cytokine-induced expression of IL-6 and IL-8 in human endothelial cells. Cytokine 8:58–65.CrossRefGoogle Scholar

Copyright information

© Feinstein Institute for Medical Research 2004

Authors and Affiliations

  • Yong Woo Lee
    • 1
  • Sung Yong Eum
    • 1
  • Kuey Chu Chen
    • 2
  • Bernhard Hennig
    • 3
  • Michal Toborek
    • 1
  1. 1.Department of Surgery/Division of NeurosurgeryUniversity of Kentucky College of MedicineLexingtonUSA
  2. 2.Molecular and Biomedical PharmacologyUniversity of Kentucky College of MedicineLexingtonUSA
  3. 3.Department of Animal SciencesUniversity of KentuckyLexingtonUSA

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