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Atherosclerosis 214 (2011) 331–337

Contents lists available at ScienceDirect

Atherosclerosis

journa l homepage: www.e lsev ier .com/ locate /a therosc leros is

pregulation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)y 15-lipoxygenase-modified LDL in endothelial cells

ngela Pirilloa,∗, Alice Reduzzia, Nicola Ferri a, Hartmut Kuhnb, Alberto Corsinia, Alberico L. Catapanoa,c

Department of Pharmacological Sciences, University of Milan, Via Balzaretti, 9, 20133 Milan, ItalyInstitute of Biochemistry, University Medicine Berlin–Charité, Berlin, GermanyIRCCS Multimedica Milan, Italy

r t i c l e i n f o

rticle history:eceived 18 June 2010eceived in revised form 18 October 2010ccepted 5 November 2010vailable online 13 November 2010

a b s t r a c t

Objective: Lectin-like oxidized LDL receptor-1 (LOX-1), the endothelial receptor for OxLDL, is believed tobe responsible for a number of OxLDL-induced effects in the endothelium.Methods and results: In the present study we showed that LDL modified by 15-lipoxygenase (15LO-LDL),a form of minimally modified lipoprotein, beside its ability to induce pro-inflammatory responses such

ey words:OX-1DL5-Lipoxygenasendothelial cells

as oxidative stress and the expression of adhesion molecules, significantly increases LOX-1 expression inendothelial cells, both at transcriptional and at protein level. Such effect is likely to be mediated by p38MAPK and NF-kB pathways. We then permanently overexpressed LOX-1 in an endothelial cell line andshowed that 15LO-LDL were a ligand for LOX-1, and that the interaction LOX-1/15LO-LDL upregulatedICAM-1 surface expression.Conclusion: Altogether these results indicate minimally modified LDL as a new inducer for LOX-1 expres-

for LO

CAM-1 sion and as a new ligand

. Introduction

Oxidation of low density lipoprotein (LDL) occurs in vivo and isnvolved in all stages of atherosclerosis [1]. When plasma lipopro-eins enter the subintimal space they can be oxidized by several

echanisms, including enzymatic and non-enzymatic pathways,hus becoming a ligand for scavenger receptors on macrophageseading to the generation of foam cells [2]. Moreover, oxidizedDL (OxLDL) can promote endothelial activation and dysfunc-ion, following the interaction between modified lipoprotein andectin-like oxidized low-density lipoprotein receptor-1 (LOX-1),he major receptor for OxLDL in endothelial cells [3]. In early humantherosclerotic lesions LOX-1 is detectable in endothelial cells; indvanced lesions LOX-1 can be also found in macrophages and inmooth muscle cells, contributing to the transformation of theseells into foam cells [4]. LOX-1 expression is up-regulated by aumber of pro-inflammatory factors such as C-reactive protein,ndothelin-1, tumor necrosis factor-alpha (TNF-�), shear stress,

ngiotensin-II and OxLDL [5,6], and in pro-atherogenic conditionsn vivo such as hyperlipidemia, hypertension and diabetes [7–9].

OxLDL are both inducers and ligands of LOX-1; a number oftudies have shown that LDL oxidized with copper up-regulate

∗ Corresponding author. Tel.: +39 02 50318293; fax: +39 02 50318386.E-mail address: angela.pirillo@guest.unimi.it (A. Pirillo).

021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2010.11.006

X-1.© 2010 Elsevier Ireland Ltd. All rights reserved.

LOX-1 expression in endothelial cells [10–12]. Among the varietyof modified LDL described in the literature, minimally modifiedLDL exhibit pro-atherogenic properties, despite minimal changesin their chemico-physical characteristics. In the present study weaimed at investigating whether other forms of modified LDL couldmodulate the expression of LOX-1 in endothelial cells. To this aim,we modified LDL with 15-lipoxygenase, an enzyme overexpressedin human and animal early atherosclerotic lesions [13,14] andinvolved in LDL modification [15–17]. We obtained a minimallymodified lipoprotein with low TBARS levels compared to copper-modified LDL, with a moderate increase in electrophoretic mobility,but capable to induce the expression of LOX-1 in endothelial cellsat both RNA and protein levels.

2. Materials and methods

2.1. Materials

Medium M199 was from Invitrogen. MEM, fetal bovineserum (FBS), penicillin–streptomycin, glutamine, TNF-�, DiO (3,3′-dioctadecyloxacarbocyanine perchlorate), 2,7-dichlorofluorescein

diacetate (DCFH-DA), k-carrageenan and SB203580 were fromSigma–Aldrich (St. Louis, MO, USA); BAY11-7085 was from Alexis;PD10 columns and ECL were from Amersham Biosciences (Upp-sala, Sweden). Endothelial cell growth factor (ECGF) was fromBoehringer Mannheim. Antibodies were purchased as follows: anti-

3 clerosis 214 (2011) 331–337

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Table 1Sequences of primers used in RT-PCR experiments.

Primer Sequence

RLP-13AForward 5′-TAG CTG CCC CAC AAA ACC-3′

Reverse 5′-TGC CGT CAA ACA CCC TTG AGA-3′

LOX-1Forward 5′-GAG AGT AGC AAA TTG TTC AGC TCC TT-3′

Reverse 5′-GCC CGA GGA AAA TAG GTA ACA GT-3′

32 A. Pirillo et al. / Atheros

OX-1 from R&Dsystem; anti-CD54 (ICAM-1) from Bio-Optica; goatnti-mouse IgG-FITC from Becton Dickinson; anti-�-actin and anti-ouse IgG peroxidase-conjugate from Sigma–Aldrich.

.2. Cell culture

Human umbilical vein endothelial cells (HUVEC) were isolatedccording to established procedures [18], cultured under stan-ard conditions in medium M-199 containing 20% FCS, heparin15 U/ml) and ECGF (20 �g/ml) (Boehringer Mannheim, Mannheim,ermany) and used within the 5th passage.

EA.hy-926 cells [19] were grown in MEM additioned of 10% FBS,% streptomycin, 1% penicillin, 2% tricine, 1% glutamine, 1% nonssential aminoacids and 1% HAT.

.3. Isolation and modification of low density lipoproteins

The use of human material in this study conforms tohe principles outlined in the Declaration of Helsinki. LDLd = 1.019–1.063 g/ml) was isolated from fresh plasma of nor-

olipidemic healthy volunteers by sequential ultracentrifugation20]. Protein content was determined by the method of Lowry’ssing BSA as standard [21]. LDL was modified at a protein con-entration of 0.2 mg/ml by exposure to 32 �g rabbit reticulocyte5-lipoxygenase/mg LDL protein at 37 ◦C for 72 h. As control, LDLas incubated at 37 ◦C for 72 h in the absence of 15-lipoxygenase.

or copper-mediated oxidation, 1.7 mg protein/ml was exposed touSO4 5 �M for 18 h [22].

Lipoprotein oxidation extent was evaluated as thehiobarbituric-acid reactive substances (TBARS) content by aolorimetric method [23]. Conjugated diene formation was mea-ured by determining the increase in absorbance at 234 nm. Thebsorbance was measured every 5 min using a Beckman 6400pectophotometer and the results were expressed as the increasen the absolute absorbance at 234 nm.

.4. Agarose gel electrophoresis

Aliquots of native and modified LDL were electrophoresed on0.8% agarose gel in 0.1 M Tris buffer (pH 8.6) for 1 h at 50 V and

tained with Sudan black.

.5. Fluorescent labeling of lipoproteins

For the lipid labeling, lipoproteins were incubated with the fluo-escent dye DiO (300 �g DiO/mg LDL protein) in PBS for 18 h at 4 ◦C,assed on a PD10 column to remove unbound DiO, then centrifuged

n a TL100 centrifuge at d = 1.063 g/ml for 2 h at 4 ◦C. DiO-labeledipoproteins were passed through a PD10 column and protein con-ent was determined by the method of Lowry.

.6. Quantitative real-time PCR (RT-PCR)

Total RNA was extracted and reverse transcribed. Three �lf cDNA were amplified by real-time quantitative polymerasehain reaction (PCR) with 1× SYBR Green universal PCR master-ix (BioRad) [24]. The sequence of the primers used for RLP13A

housekeeping gene), LOX-1 and ICAM-1 amplification are reportedn Table 1. Each sample was analyzed in duplicate using theQTM-Cycler (BioRad). For quantification, the target genes were nor-

alized to the RLP13A content.

.7. Immunoblotting

To analyze the expression of LOX-1, cells were lysed with auffer containing 2% SDS, 62.5 mM Tris, 50 mM DTT, 1 mM PMSF,

ICAM-1Forward 5′-GCC GGC CAG CTT ATA CAC AA-3′

Reverse 5′-CAA TCC CTC TCG TCC AGT CG-3′

5 �g/ml aprotinin and sonicated; cell proteins were separated ona 10% SDS-PAGE, then transferred onto a nitrocellulose mem-brane. Protein expression was analyzed by immunoblotting usinga mouse anti-human LOX-1 antibody (1:1000); a mouse anti-�-actin antibody (1:1000) was used to normalize the protein loading.After incubation with an anti-mouse IgG peroxidase-conjugatedas secondary antibody, immunocomplexes were detected by ECLfollowed by autoradiography.

2.8. Cell-association studies

For cell-association studies, cells were incubated at 37 ◦C for 1 hwith the indicated concentrations of native or 15-LO-modified LDLlabeled with DiO. Cells were then washed thrice with cold PBS,detached by scraping or trypsinization, fixed in 1% paraformalde-hyde and immediately subjected to fluorescence flow cytometryusing a FACScan (Becton Dickinson). For each sample 10,000 eventswere analyzed; data were processed using the CellQuest program(Becton Dickinson).

2.9. Measurement of ICAM-1 surface expression

HUVEC were incubated for 18 h in the presence of native or 15-LO-modified LDL (50 �g/ml). At the end of the incubation, cells wereharvested by trypsinization, washed in PBS–BSA (1%) and incu-bated with anti-CD54 (ICAM-1) monoclonal antibody and then withgoat anti-mouse IgG-FITC, as described [25]. After washing, anti-gen expression was measured by flow cytometry (FACScan, BectonDickinson). Isotype controls were obtained by incubating cells withgoat anti-mouse IgG-FITC to determine non-specific fluorescence.Cells were gated and data were obtained from fluorescence chan-nels in a logarithmic mode. A total of 10,000 events were analyzed;data were processed using the CellQuest program.

2.10. Measurement of ROS generation

The generation of intracellular reactive oxygen species (ROS)was measured by monitoring the fluorescence of DCFH-DA, aprobe sensitive to the oxidation status. Cells were pre-incubatedwith 50 �g/ml DCFH-DA for 1 h, then exposed to native or 15-LO-modified LDL for a further 1 h. Cells were then harvested bytrypsinization and resuspended in PBS/BSA 1% for immediate deter-mination of oxidative stress by flow cytometry.

2.11. Generation and characterization of humanLOX1-overexpressing EAhy cells

The retroviral expression plasmid encoding for human LOX-1

was kindly provided by Dr. Elaine Raines (University of Wash-ington, Seattle, WA), constructed using the pBM-IRES-PURO,expressing the puromycin resistance gene as a selectable secondcistron gene, generated from the original pBM-IRES-EGFP, gener-ously provided by G.P. Nolan (Stanford University, Stanford, CA,

A. Pirillo et al. / Atherosclerosis 214 (2011) 331–337 333

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ig. 1. Characterization of LDL modified by 15-lipoxygenase. (A) Native LDL, 15Lvaluate electrophoretic mobility. (B) TBARS content of native and modified LDL. (DL oxidation. (D and E) Interaction of native or modified LDL with J774 (D) and HUt 37 ◦C. Cell-associated fluorescence was evaluated by flow cytometry. Data are mDL.

SA). Retroviral infection of EAhy was performed as previouslyescribed [26].

LOX-1 overexpression was assessed as mRNA and as protein.RNA expression was evaluated by real-time PCR; total protein

xpression was evaluated by western blotting as described above;urface protein expression was analyzed by flow cytometry usingmonoclonal antibody anti-LOX-1 followed by a goat anti-mouse

gG-FITC.To characterize LOX-1 overexpression, EAhy and EAhy-LOX-1

ere incubated with increasing amount of DiO-OxLDL for 1 h at7 ◦C. Cells were then washed thrice with cold PBS, detached bycraping, fixed in 1% paraformaldehyde and immediately subjectedo fluorescence flow cytometry. In some experiments, cells werere-incubated with k-carrageenan (250 �g/ml) for 1 h before addi-ion of DiO-OxLDL [27].

.12. Statistical analysis

Values are expressed as means ± SD. The statistical significancef the differences between groups was determined by the Student’s-test and values of P < 0.05 were considered to be significant.

. Results

.1. Modification of LDL with 15LO

The incubation of LDL with 15LO slightly increased the elec-rophoretic mobility on agarose gel compared to native LDLFig. 1A), while the change of electrophoretic mobility was, as

and Cu2+-LDL were run on a 0.8% agarose gel and stained with Sudan black toe course of conjugated diene formation during Cu2+-mediated or 15LO-mediated

E). J774 or HUVEC were incubated with DiO-labeled lipoproteins (10 �g/ml) for 1 hSD of 3 independent experiments performed in duplicate. *P < 0.05, **P < 0.0005 vs.

expected, much higher for Cu2+-modified LDL. Accordingly, theenzymatic modification moderately increased the TBARS contentin 15LO-LDL that resulted considerably higher in LDL modified bycopper (Fig. 1B). The Cu2+- and 15LO-mediated oxidation of LDLwas evaluated also as formation of conjugated diene at 234 nm. Thelag-time was about 43 min for Cu2+-oxidized LDL and 247 min for15LO-LDL (Fig. 1C); despite the different lag-time and the differentTBARS content, the levels of reached conjugated diene were similar.

Besides the low TBARS content, an additional property of mini-mally modified LDL is their inability to bind macrophage scavengerreceptors [28,29]. Accordingly, we found that Cu2+-oxidized LDLefficiently interacted with macrophages, while 15LO-LDL poorlybound to these cells (Fig. 1D). Cell-association studies performedwith endothelial cells, however, revealed that OxLDL did not inter-act efficiently with this cell type, compared to native LDL, andsimilarly the association of 15LO-LDL with endothelial cells wassignificantly lower compared to native LDL (Fig. 1E).

3.2. Increased expression of ICAM-1 in endothelial cells incubatedwith 15LO-LDL

HUVEC were stimulated with 50 �g/ml LDL, 15LO-LDL or Cu-LDL for 6 h and the expression of ICAM-1 mRNA was investigated

by real-time PCR. LDL did not alter significantly ICAM-1 mRNA(Fig. 2A). The incubation of endothelial cells with 15LO-LDL signif-icantly increased the expression of ICAM-1 mRNA by almost 4-foldcompared to control cells (Fig. 2A); the induction was similar tothat obtained with Cu-LDL.

334 A. Pirillo et al. / Atherosclerosis 214 (2011) 331–337m

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Fig. 3. Reactive oxygen species production in endothelial cells. HUVEC were pre-incubated with DCFH-DA (50 �g/ml) for 1 h, then incubated 1 h with 50 �g/ml LDL,15LO-LDL or Cu-LDL. Cells were harvested for immediate determination of oxida-tive stress by flow cytometry. Results are given as mean ± SD from 7 independentexperiments. *P < 0.0005 vs. LDL.

ig. 2. Upregulation of ICAM-1 expression in endothelial cells incubated with 15LO-DL. HUVEC were incubated with LDL, 15LO-LDL or Cu-LDL (50 �g/ml) for 6 h (A) or8 h (B). At the end of the incubation gene expression by real time PCR (A) and cellurface expression by flow cytometry (B) were evaluated. Data are means ± SD of 4A) and 8 (B) independent experiments. *P < 0.05, **P < 0.00005 vs. LDL.

We then investigated the expression of ICAM-1 at protein level:he incubation of endothelial cells with LDL did not change themount of ICAM-1 at cell surface compared to control cells, whilehe exposure to 15LO-LDL, as well as Cu-LDL, significantly increasedhe expression of ICAM-1 at cell surface (Fig. 2B).

.3. Effect of LDL modification by 15LO on the oxidative stresstatus in HUVEC

As the generation of reactive oxygen species (ROS) is a key mech-nism in the induction of adhesion molecules in endothelial cellsy different stimuli [30], we investigated the effect of 15LO-LDLn the oxidative stress status in HUVEC. While LDL did not stimu-ate the generation of ROS compared to untreated cells, 15LO-LDLignificantly increased oxidative stress (Fig. 3), even if at a lowerxtent compared to Cu-LDL.

.4. LOX-1 expression in HUVEC exposed to 15LO-LDL

To study the effect of 15LO-LDL on the expression of LOX-1 inndothelial cells, HUVEC were exposed to LDL, 15LO-LDL or Cu-DL for 18 h and the expression of LOX-1 mRNA was investigatedy real-time PCR. Native LDL did not affect the expression of LOX-1RNA, while 15LO-LDL significantly increased the transcription of

OX-1 gene (Fig. 4A). As expected, Cu-LDL significantly increased

OX-1 mRNA expression.

Western blotting analysis of cell lysates confirmed a signifi-antly increased level of LOX-1 protein in endothelial cells exposedo either 15LO-LDL or Cu-LDL, compared to cells incubated withDL (Fig. 4B).

Fig. 4. Upregulation of LOX-1 expression in endothelial cells exposed to 15LO-LDL.HUVEC were incubated with LDL, 15LO-LDL or Cu-LDL (50 �g/ml) for 18 h (A) or 24 h(B). At the end of the incubation gene expression by real time PCR (A) and proteinexpression by western blotting (B) were evaluated. Data are means ± SD of 7 (A) and3 (B) independent experiments. *P < 0.005, **P < 0.0005 vs. LDL.

clerosis 214 (2011) 331–337 335

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Fig. 5. Inhibition of 15LO-LDL-induced LOX-1 mRNA expression by p38 and NF-kB inhibitors. HUVEC were pre-incubated with BAY11-7085 (5 �M) or SB203580(20 �M) for 1 h, then incubated with 15LO-LDL (50 �g/ml) for 18 h. LOX-1 expressionwas evaluated by real time PCR. Results are given as mean ± SD from 4 independent

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A. Pirillo et al. / Atheros

.5. Involvement of p38 and NF-kB in 15LO-LDL-mediated LOX-1xpression

Endothelial cells were pre-incubated with p38 inhibitorB203580 (20 �M) or NF-kB inhibitor BAY11-7085 (5 �M) for 1 hefore addition of 15LO-LDL. Both inhibitors significantly reducedhe mRNA levels of LOX-1 in endothelial cells incubated with 15LO-DL (Fig. 5), suggesting the involvement of p38 MAPK and NF-kBctivation in the induction of LOX-1 gene transcription by 15LO-DL.

.6. Generation and characterization of an endothelial cell lineverexpressing LOX-1

Infection of EAhy cells with plasmid encoding for human LOX1ignificantly increased LOX-1 expression both at transcriptionalnd at protein level (Fig. 6A and B); only a fraction of LOX-1 pro-ein was localized at the plasma membrane surface (Fig. 6C). Thencubation of LOX-1-overexpressing cells with DiO-OxLDL signifi-antly increased the association with labeled lipoprotein, compared

o EAhy wild type cells (Fig. 6C); no changes were observed in thessociation of cells with native LDL (data not shown). Pre-treatmentf EAhy-LOX-1 cells with k-carrageenan, a well characterizedOX-1 ligand, significantly inhibited OxLDL association to LOX-1-verexpressing endothelial cells (Fig. 6D).

ig. 6. Establishment of an endothelial cell line overexpressing LOX-1. EAhy-926 cells wxpression of LOX-1 was evaluated at mRNA level by real-time PCR (A) and at protein levith EAhy-926 wild type or EAhy-926 overexpressing LOX-1 receptor. (E) EAhy and EAhyxLDL for 1 h. Cell-association was determined by flow cytometry. *P < 0,005, **P < 0.0005

experiments. *P < 0.001, **P < 0.0005 vs. 15LO-LDL.

ere infected with a retroviral expression plasmid encoding for human LOX1. Over-el by western blotting (B) and by flow cytometry (C). (D) Cell-association of OxLDL-LOX-1 were preincubated with 250 �g/ml k-carrageenan for 1 h, then exposed tovs. EAhy.

336 A. Pirillo et al. / AtherosclerosIC

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.7. Modulation of ICAM-1 expression by 15LO-LDL throughOX-1

As first, we compared cell-association of DiO-labeled 15LO-DL to EAhy and EAhy-LOX-1 cells. We found that overexpressionf LOX-1 in endothelial cells significantly increased the cell-ssociation of 15LO-LDL, compared to control cells (Fig. 7A). Thene evaluated ICAM-1 surface expression in EAhy and in EAhy-

OX-1 cells exposed to 50 �g/ml LDL, 15LO-LDL or Cu-LDL. Inreliminary experiments, we found that EAhy were not a goododel for adhesion molecule induction studies, as ICAM-1 wasarkedly upregulated in this cell line only in the presence of strong

ro-inflammatory stimuli such as TNF�, while the incubation withodified lipoproteins only slightly increased the expression of

dhesion molecules compared to HUVEC (data not shown). How-ver, the overexpression of LOX-1 markedly increased the amountf ICAM-1 expressed at cell surface in cells incubated with 15LO-DL or Cu-LDL (Fig. 7B), indicating that 15LO-LDL can mediate itsffects through LOX-1 activation.

. Discussion

One of the earliest events in atherogenesis is the endothelialctivation that can be triggered by OxLDL through a variety ofechanisms, such as the generation of reactive oxygen species, the

is 214 (2011) 331–337

upregulation of adhesion molecules [31] and the downregulationof eNOS activity [32]. The identification of lectin-like oxidized LDLreceptor-1 (LOX-1) in endothelial cells as the receptor for OxLDLprovided further insight into the effects of OxLDL on the vascu-lar endothelium. LOX-1 is a scavenger receptor that contributesto all stages of atherosclerosis; its expression is upregulated bymany factors relevant to atherosclerosis such as OxLDL, tumornecrosis factor, angiotensin II, C reactive protein, high glucose [6].LOX-1 is also the major receptor responsible for OxLDL binding,internalization and degradation in endothelial cells [33], where itmediates a number of pro-atherogenic effects triggered by OxLDL[34].

OxLDL represent a heterogeneous group of lipoproteins that canmodulate cellular functions on the basis of their degree of modifica-tion and on the type of cells involved. Among the enzymes whoseexpression is up regulated in the cells of the arterial wall underpro-inflammatory conditions 15-lipoxygenase, because of its abil-ity to modify LDL, has received particular attention [35–37]. Manystudies have, in fact, documented the presence of 15-lipoxygenasein atherosclerotic lesions and its colocalization with epitopes ofoxidized LDL [13,14]. Furthermore, 15-lipoxygenase modifies HDLin vitro, thus impairing its role in the cholesterol efflux process[38] and its anti-inflammatory activity [25]. In the present studywe show that in vitro modification of LDL with 15-lipoxygenase,results in a lipoprotein with several of the characteristics of min-imally modified LDL, such as a slight increase in the relativeelectrophoretic mobility, a moderate increase in the TBARS con-tent, and the failure to be recognized by macrophage scavengerreceptors. Despite the minimal modification, 15LO-LDL did notinteract with endothelial cells as native LDL did, suggesting thatthe modification mediated by 15LO reduced the ability of 15LO-LDLto be recognized by LDL receptor as well. Furthermore, 15LO-LDLinduced the expression of adhesion molecules in endothelial cells,suggesting that the enzymatic modification of LDL, though mini-mal, conferred pro-inflammatory characteristics to the lipoprotein.Interestingly, 15LO-LDL could also significantly induce the expres-sion of LOX-1 in endothelial cells at both mRNA and protein level,providing a role for minimally modified LDL in the induction ofLOX-1.

OxLDL–LOX-1 interaction induces the generation of ROS andstimulates pro-inflammatory gene expression [39], thus inducingendothelial dysfunction. Moreover, LOX-1 levels are elevated byOxLDL binding [40], suggesting a positive-feedback loops enhanc-ing vascular dysfunction. The generation of an endothelial cellline overexpressing LOX-1 helped us to show that 15LO-LDLnot only were effective at increasing endothelial expression ofLOX-1, but also represented a ligand for LOX-1. In fact the bind-ing of 15LO-LDL was significantly higher in cells overexpressingLOX-1 compared to wild type cells; moreover, the incubationof endothelial cells overexpressing LOX-1 with 15LO-LDL signifi-cantly increased ICAM-1 expression compared to wild type cells.Altogether these results confirm a pro-inflammatory properties ofenzymatically minimally modified LDL that, perhaps is further sus-tained by the ability to induce LOX-1 expression and to inducea pro-inflammatory response in endothelial cells through LOX-1interaction.

In summary the main finding of the present study is that avery low degree of modification obtained by LDL exposure to 15LOgenerates a lipoprotein with pro-inflammatory features, able toinduce LOX-1 expression in endothelial cells and capable of pro-moting endothelial cell activation through interaction with LOX-1.These results suggested that during the early phases of athero-genesis, when 15LO is overexpressed by activated macrophages,

the enzyme-mediated modification of LDL might initiate endothe-lial cell dysfunction and sustain it by promoting LOX-1 receptorexpression.

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[39] Dunn S, Vohra RS, Murphy JE, et al. The lectin-like oxidized low-density-lipoprotein receptor: a pro-inflammatory factor in vascular disease. Biochem J

A. Pirillo et al. / Atheros

isclosures

The authors have no financial conflict of interest.

cknowledgements

This work was supported by SISA (Società Italiana per lo Studioell’Aterosclerosi, Lombardia section).

eferences

[1] Stocker R, Keaney Jr JF. Role of oxidative modifications in atherosclerosis. Phys-iol Rev 2004;84:1381–478.

[2] Boullier A, Bird DA, Chang MK, et al. Scavenger receptors, oxidized LDL, andatherosclerosis. Ann N Y Acad Sci 2001;947:214–22 [discussion 22–3].

[3] Sakurai K, Sawamura T. Stress and vascular responses: endothelial dysfunctionvia lectin-like oxidized low-density lipoprotein receptor-1: close relationshipswith oxidative stress. J Pharmacol Sci 2003;91:182–6.

[4] Kataoka H, Kume N, Miyamoto S, et al. Expression of lectinlike oxidized low-density lipoprotein receptor-1 in human atherosclerotic lesions. Circulation1999;99:3110–7.

[5] Kume N, Kita T. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)in atherogenesis. Trends Cardiovasc Med 2001;11:22–5.

[6] Vohra RS, Murphy JE, Walker JH, Ponnambalam S, Homer-Vanniasinkam S.Atherosclerosis and the Lectin-like OXidized low-density lipoprotein scavengerreceptor. Trends Cardiovasc Med 2006;16:60–4.

[7] Chen M, Kakutani M, Minami M, et al. Increased expression of lectin-likeoxidized low density lipoprotein receptor-1 in initial atherosclerotic lesionsof Watanabe heritable hyperlipidemic rabbits. Arterioscler Thromb Vasc Biol2000;20:1107–15.

[8] Nagase M, Hirose S, Sawamura T, Masaki T, Fujita T. Enhanced expression ofendothelial oxidized low-density lipoprotein receptor (LOX-1) in hypertensiverats. Biochem Biophys Res Commun 1997;237:496–8.

[9] Chen M, Nagase M, Fujita T, et al. Diabetes enhances lectin-like oxidized LDLreceptor-1 (LOX-1) expression in the vascular endothelium: possible role ofLOX-1 ligand and AGE. Biochem Biophys Res Commun 2001;287:962–8.

10] Li D, Mehta JL. Antisense to LOX-1 inhibits oxidized LDL-mediated upregula-tion of monocyte chemoattractant protein-1 and monocyte adhesion to humancoronary artery endothelial cells. Circulation 2000;101:2889–95.

11] Hoshikawa H, Sawamura T, Kakutani M, et al. High affinity binding of oxidizedLDL to mouse lectin-like oxidized LDL receptor (LOX-1). Biochem Biophys ResCommun 1998;245:841–6.

12] Aoyama T, Fujiwara H, Masaki T, Sawamura T. Induction of lectin-like oxi-dized LDL receptor by oxidized LDL and lysophosphatidylcholine in culturedendothelial cells. J Mol Cell Cardiol 1999;31:2101–14.

13] Yla-Herttuala S, Rosenfeld ME, Parthasarathy S, et al. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low densitylipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl AcadSci U S A 1990;87:6959–63.

14] Yla-Herttuala S, Rosenfeld ME, Parthasarathy S, et al. Gene expression inmacrophage-rich human atherosclerotic lesions 15-lipoxygenase and acetyllow density lipoprotein receptor messenger RNA colocalize with oxidationspecific lipid–protein adducts. J Clin Invest 1991;87:1146–52.

15] Harats D, Shaish A, George J, et al. Overexpression of 15-lipoxygenase in vas-cular endothelium accelerates early atherosclerosis in LDL receptor-deficientmice. Arterioscler Thromb Vasc Biol 2000;20:2100–5.

16] Huo Y, Zhao L, Hyman MC, et al. Critical role of macrophage 12/15-lipoxygenase for atherosclerosis in apolipoprotein E-deficient mice. Circulation2004;110:2024–31.

17] Bocan TM, Rosebury WS, Mueller SB, et al. A specific 15-lipoxygenase

inhibitor limits the progression and monocyte-macrophage enrichment ofhypercholesterolemia-induced atherosclerosis in the rabbit. Atherosclerosis1998;136:203–16.

18] Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cellsderived from umbilical veins. Identification by morphologic and immunologiccriteria. J Clin Invest 1973;52:2745–56.

[

is 214 (2011) 331–337 337

19] Edgell CJ, McDonald CC, Graham JB. Permanent cell line expressing human fac-tor VIII-related antigen established by hybridization. Proc Natl Acad Sci U S A1983;80:3734–7.

20] Havel RJ, Eder HA, Bragdon JH. The distribution and chemical compositionof ultracentrifugally separated lipoproteins in human serum. J Clin Invest1955;34:1345–53.

21] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with theFolin phenol reagent. J Biol Chem 1951;193:265–75.

22] Cominacini L, Pasini AF, Garbin U, et al. Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 in endothelial cells induces the activationof NF-kappaB through an increased production of intracellular reactive oxygenspecies. J Biol Chem 2000;275:12633–8.

23] Schuh J, Fairclough Jr GF, Haschemeyer RH. Oxygen-mediated heterogeneity ofapo-low-density lipoprotein. Proc Natl Acad Sci U S A 1978;75:3173–7.

24] Norata GD, Tibolla G, Seccomandi PM, Poletti A, Catapano AL. Dihydrotestos-terone decreases tumor necrosis factor-alpha and lipopolysaccharide-inducedinflammatory response in human endothelial cells. J Clin Endocrinol Metab2006;91:546–54.

25] Pirillo A, Uboldi P, Bolego C, Kuhn H, Catapano AL. The 15-lipoxygenase-modified high density lipoproteins 3 fail to inhibit the TNF-alpha-induced inflammatory response in human endothelial cells. J Immunol2008;181:2821–30.

26] Garton KJ, Ferri N, Raines EW. Efficient expression of exogenous genes inprimary vascular cells using IRES-based retroviral vectors. Biotechniques2002;32:830, 2, 4 passim.

27] Mehta JL, Li DY. Identification and autoregulation of receptor for OX-LDL in cul-tured human coronary artery endothelial cells. Biochem Biophys Res Commun1998;248:511–4.

28] Berliner JA, Territo MC, Sevanian A, et al. Minimally modified low den-sity lipoprotein stimulates monocyte endothelial interactions. J Clin Invest1990;85:1260–6.

29] Hofnagel O, Luechtenborg B, Weissen-Plenz G, Robenek H. Statins and foamcell formation: impact on LDL oxidation and uptake of oxidized lipoproteinsvia scavenger receptors. Biochim Biophys Acta 2007;1771:1117–24.

30] Weber C, Erl W, Pietsch A, et al. Antioxidants inhibit monocyte adhesion bysuppressing nuclear factor-kappa B mobilization and induction of vascularcell adhesion molecule-1 in endothelial cells stimulated to generate radicals.Arterioscler Thromb 1994;14:1665–73.

31] Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modifiedlow density lipoproteins: a potential role in recruitment and retention ofmonocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A1987;84:2995–8.

32] Blair A, Shaul PW, Yuhanna IS, Conrad PA, Smart EJ. Oxidized lowdensity lipoprotein displaces endothelial nitric-oxide synthase (eNOS)from plasmalemmal caveolae and impairs eNOS activation. J Biol Chem1999;274:32512–9.

33] Sawamura T, Kume N, Aoyama T, et al. An endothelial receptor for oxidizedlow-density lipoprotein. Nature 1997;386:73–7.

34] Navarra T, Del Turco S, Berti S, Basta G. The lectin-like oxidized low-densitylipoprotein receptor-1 and its soluble form: cardiovascular implications. JAtheroscler Thromb 2009.

35] Sigal E, Laughton CW, Mulkins MA. Oxidation, lipoxygenase, and atherogenesis.Ann N Y Acad Sci 1994;714:211–24.

36] Feinmark SJ, Cornicelli JA. Is there a role for 15-lipoxygenase in atherogenesis?Biochem Pharmacol 1997;54:953–9.

37] Funk CD, Cyrus T. 12/15-lipoxygenase, oxidative modification of LDL andatherogenesis. Trends Cardiovasc Med 2001;11:116–24.

38] Pirillo A, Uboldi P, Kuhn H, Catapano AL. 15-Lipoxygenase-mediated mod-ification of high-density lipoproteins impairs SR-BI- and ABCA1-dependentcholesterol efflux from macrophages. Biochim Biophys Acta 2006;1761:292–300.

2008;409:349–55.40] Aoyama T, Sawamura T, Furutani Y, et al. Structure chromosomal assignment

of the human lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1)gene. Biochem J 1999;339(Pt 1):177–84.