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Cardiovascular Research Advance Access first published online on September 18, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn256
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion

Jakub Rohlena1,{dagger}, Oscar L. Volger2, Jaap D. van Buul3, Liesbeth H.P. Hekking4, Janine M. van Gils3, Peter I. Bonta1, Ruud D. Fontijn2, Jan Andries Post4, Peter L. Hordijk3 and Anton J.G. Horrevoets2,*

1 Department of Medical Biochemistry, Academic Medical Centre, Amsterdam, The Netherlands
2 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Room FG-B244, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
3 Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, Amsterdam, The Netherlands
4 Department of Cell Biology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands

* Corresponding author. Tel: +31 20 4448161; fax: +31 20 4448081. E-mail address: aj.horrevoets{at}vumc.nl

Received 29 April 2008; revised 29 August 2008; accepted 15 September 2008

Time for primary review: 23 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 Supplementary material
 References
 
Aims: In a recent report, we established at the genome-wide level those genes that are specifically upregulated in the endothelium of atherosclerotic plaques in human arteries. As the transcriptome data revealed that mRNA for the tetraspanin family member CD81 is significantly and specifically upregulated in the endothelium overlying early atheroma, we set out to validate these results on the protein level, and investigate the functional consequences of CD81 upregulation.

Methods and results: Immunohistochemical analysis in an independent set of donor arteries verified in the endothelium of early human atherosclerotic lesions the enhanced expression of CD81, which appears oxidative stress-dependent. Using lentiviral overexpression and silencing in human umbilical endothelial cells, we established in an in vitro flow adhesion assay that elevated endothelial CD81 is associated with increased monocyte adhesion to non-activated CD81-transduced endothelial cells, approaching the levels normally only attained after tumour necrosis factor {alpha} stimulation. The CD81 effect was dependent on both intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as it was abolished in the presence of a mixture of anti-ICAM-1 and anti-VCAM-1 antibodies. Flow cytometry revealed that increased CD81 levels did not increase total ICAM-1 and VCAM-1 surface expression. Instead, it concentrated the available adhesion molecules into membrane clusters, as indicated by confocal and electron microscopy. CD81 also colocalized with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes.

Conclusion: Endothelial CD81 upregulated in early human atheroma has the potential to play a crucial role in the initial stages of atherosclerotic plaque formation by increasing monocyte adhesion prior to the full-blown inflammatory response.

KEYWORDS Endothelium; Atherosclerosis; Adhesion molecules


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 Supplementary material
 References
 
Atherosclerosis is a pathogenic condition of the arterial vessel wall, characterized by lipid deposition, leukocyte infiltration, and intimal thickening.1 Besides the well-known systemic risk factors, such as smoking, high blood pressure, and high blood cholesterol, local conditions at the vessel wall also play an important role in the progression of atherosclerosis. This is exemplified by the distinct focal pattern of lesion formation along the vascular tree.2 As these local influences are as yet not fully understood, it is imperative that we dissect molecular processes within the lesion sites, and how they differ from those in the neighbouring vessel wall that remains lesion-free. One of the major determinants of the vessel wall fitness is the laminar blood flow conferring the shear stress force onto the endothelial cells lining the artery, which alters the activity of KLF2 and ATF2 transcription factors, resulting in quiescent and healthy endothelium.3–7 However, as other molecular factors may also play a role, we recently established the specific gene expression in endothelium overlaying human atherosclerotic lesions, to identify genes with a potential to modulate intimal accumulation of macrophages.8,9

One of the genes we identified as candidate by this transcriptome analysis is CD81 (also known as TAPA-1), a member of the tetraspanin superfamily of transmembrane proteins.10,11 Typical of tetraspanins is the propensity to form lipid-raft-like membrane microdomains, assembled through interaction between individual tetraspanin molecules and other membrane proteins, such as integrins and adhesion molecules of the immunoglobulin superfamily. These microdomains contribute to the enhancement of various cellular processes, including receptor signalling and cellular adhesion.11–13 CD81 is most extensively studied in leukocytes where it facilitates integrin-mediated adhesion to VCAM-1 under flow,14 but is also expressed by endothelial cells.15 In the present study, we show that CD81 is upregulated in the endothelium of early atherosclerotic lesions, and that it has the potential to substantially enhance monocyte adhesion via relocalization and increased clustering of endothelial adhesion molecules ICAM-1 and VCAM-1.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 Supplementary material
 References
 
2.1 Immunohistochemistry
The analysis of CD81 expression in human arteries was done as previously described.8 Human large arteries were collected post-mortem after disease in compliance with the Academic Medical Center institutional guidelines, in full compliance with the principles and ethical considerations of the Declaration of Helsinki (1989). The atherosclerotic plaques were classified according to Virmani et al.16 Monoclonal antibodies for CD81 detection were either clone 1D6 (Serotec, Oxford, UK) for donors D12 en D1316, or clone 1.3.3.2 [EC] 2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for the rest of the donors, depending on the type of tissue fixation. Donor data can be found in Volger et al.8 The remaining two donors not described there were as follows: D3312, male, 48 years, cause of death unknown, descending aorta; D1316, female, 76 years, cause of death unknown, bifurcating iliac artery.

2.2 Cell culture
Human umbilical endothelial cells (HUVEC) were isolated and cultured as described17–19 in Medium 199 (Invitrogen, Paisly, UK), or in EGM-2 (Cambrex, Verviers, Belgium) according to manufacturer's instructions, using 8% (v/v) foetal calf serum and penicillin/streptomycin, and used at passage 1 or 2. HL60 cell line was maintained in Optimem medium (Invitrogen) supplemented with 5% foetal calf serum, and differentiated by 1.3% DMSO (Sigma, St Louis, MO, USA). Primary monocytes were isolated from fresh whole blood by means of magnetic-activated cell sorting using CD14+ microbeads or the Monocyte isolation kit according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).

2.3 Stimulation experiments
Confluent HUVEC in fibronectin coated 12-well plates were grown overnight in M199 medium supplemented with 1% human serum albumin (Sanquin Reagens, Amsterdam, The Netherlands) and transferrin-insulin-selenite growth supplement (Sigma), and were next stimulated for 24 h with 50 ng/mL TNF{alpha}, 10 ng/mL IL-1β, 10 ng/mL Angiopoietin-1, 40 ng/mL VEGF, 10 ng/mL TGFβ (all R&D Systems, Abingdon, UK), 100 µM Histamin (Sigma), or medium from differentiated THP1 monocytic cell line cultured in the presence of oxLDL (Intracel, Frederick, MD, USA) for 24 h, or with 50 µM tert-butyl hydrogen peroxide (Sigma) or 10 µM phenazine methosulfate (PMS, Sigma) for 16 h. Stretch was applied for 24 h,4 and laminar flow for 24 h or 6 days.7,20

2.4 Lentiviral overexpression and silencing
Human CD81 cDNA and CD81-specific short-hairpin RNA (shRNA) directed against a published target sequence21 were cloned into lentiviral vectors, and lentiviral particles were produced as previously described.4,18 Lentiviral particles carrying overexpression vector lacking CD81 cDNA (Mock) or containing firefly luciferase-specific shRNA (Control si) were used as controls.20 All constructs including the controls express GFP, from an IRES element in the overexpression vectors, or from an independent CMV promoter in shRNA vectors.

2.5 Real-time RT–PCR
Real-time RT–PCR was performed as previously described.7

2.6 Monocyte adhesion and rolling
Lentivirally transduced HUVEC were grown on gelatin- and fibronectin-coated glass coverslips and grown to confluency (4–5 days). The monolayers were stimulated or not by 10 ng/mL TNF{alpha} for 16 h, and monocyte adhesion or rolling assay was performed as described using 2 x 106/mL suspension of freshly isolated primary human monocytes at a flow rate of 0.8 dyn/cm2. The adhesion or rolling were followed between 2 and 4 min of perfusion, recording between 15 and 20 random fields per coverslip and evaluating results as described,22 measuring the percentage of rolling monocytes or the amount of immobile, firmly adhered monocytes at the focus plane corresponding to the apical side of the endothelial layer using phase contrast.22 This procedure was independently performed for each coverslip and was considered one independent measurement. For blocking experiments, the monolayers were pre-treated for 30 min with the following monoclonal antibodies diluted 1/20 in the growth medium: anti-VCAM-1 (clone BBIG-V1 4B2, R&D systems), anti-ICAM-1 (clone 84H10, Beckman Coulter, Miami, FL, USA), and the negative control anti-PECAM-1 (clone CLB-HEC/75, Sanquin Reagents).

2.7 Flow cytometry
The lentivirally transduced HUVEC were split and grown to confluency. The cells were either stimulated or not by 10 ng/mL TNF{alpha} (R&D Systems) for 16 h, resuspended by trypsin, stained with indicated antibodies, and flow cytometry analysis was performed on the Facscalibur instrument (BD Biosciences, San Jose, CA, USA). Detection was as follows. Monoclonal anti-CD81 (Serotec), anti-ICAM-1 (Sanquin Reagents), and anti VCAM-1 (Southern Biotech, Birmingham, AL, USA) in 1:20 dilution were unlabelled, and were detected by a secondary Goat-anti-mouse PE labelled antibody (1:50 dilution, BD Biosciences).

2.8 Immunofluorescence and confocal microscopy
Lentivirally transduced HUVEC were grown on gelatin- and fibronectin-coated glass coverslips to confluency (4–5 days), after which the cells were stimulated or not with 10 ng/mL TNF{alpha} for 16 h. The cells were washed, fixed, and immunostained with primary monoclonal antibodies against the V5 tag (Invitrogen), ICAM-1 (R&D Systems), or polyclonal antibody against VE-cadherin (Cayman Chemicals, Ann Arbor, MI, USA) as described previously.23 Subsequent visualization was performed with Alexa-conjugated secondary antibodies (Invitrogen). For CD81/ICAM-1 colocalization experiments, V5 tag was detected by fluorescein-conjugated anti-V5 antibody (Invitrogen). F-actin was visualized with fluorescently labelled Phalloidin (Invitrogen). The images in Figure 2 were recorded with Zeiss Axioplan 2 microscope (Zeis, Oberkochen, Germany) and Coolsnap HQ digital camera (Roper Scientific, Ottobrunn, Germany), others with a ZEISS LSM510 confocal microscopy module mounted onto the Axiovert 200 inverted microscope (Zeis) with appropriate filter settings. Crosstalk between the different channels was avoided by the use of sequential scanning. All images were processed with Adobe Photoshop CS2 software.

2.9 Electron microscopy
Lentivirally transduced HUVEC were grown to confluency on fibronectin-coated Thermanox slides (Nunc, Roskilde, Denmark). After 5 days, the cells were fixed with 1% paraformaldehyde overnight and labelled with a mixture of primary antibodies against CD81 (Serotec) and ICAM-1 (Ab7815, Abcam, Cambridge, UK) in 0.5% CWF+0.1% BSA-c (Aurion, Wageningen, The Netherlands) in PBS for 1 h. Visualization was performed with a mixture of gold conjugated secondary antibodies (goat anti mouse 6 nm to detect CD81 and goat anti rabbit 10 nm for ICAM-1, Aurion). The cells were then fixed with a mixture of 2% paraformaldehyde, 2.5% glutaraldehyde, 0.025% CaCl2·2H2O, 0.05% MgCL2·6H2O, and 0.1 M Na-cacodylate pH 7.4 overnight, and dehydrated with alcohol and propylene oxide. No OsO4 post-fixation was performed. After dehydration, the cells were embedded in Epon. Thin sections (60 nm) were stained in a saturated solution of uranyl acetate in 70% methanol followed by Reynold's lead citrate. The sections were examined using a Technai 12 EM (FEI Co., Eindhoven, The Netherlands), equipped with a side-mounted Megaview II camera (SIS, Muenster, Germany).

2.10 Statistical analysis
Data represent means plus and minus standard error of the mean, and were analysed in Microsoft Excel using unpaired Student's t-test.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 Supplementary material
 References
 
3.1 CD81 is upregulated in early human atherosclerotic lesions
Recently we performed an extensive genomic study to compare the expression profiles of endothelial genes in the endothelium of atherosclerotic plaques and that of unaffected areas of the vessel wall of human arteries from the same donor.8 Analysis of microarray transcriptome data identified CD81, a member of the tetraspanin superfamily, as a specific marker discriminating early, inflamed lesions from endothelial cells overlying unaffected vessel wall, both by FDR-corrected t-testing (cf. Volger et al.,8 supplement 2A, P < 10–4), and Gene Set Enrichment Analysis approaches (cf. Volger et al.,8 supplement 4C, rank 5). For advanced lesions, however, CD81 was not significantly increased. We now performed an immunohistochemical validation on an independent set of donor arteries, showing that CD81 protein was present at increased levels in the endothelium over the early endothelial lesions (six out of seven tested), whereas it was absent (0 out of 12) in the healthy endothelium of the arterial wall (Figures 1A and Supplementary material online, Figure S1A). In contrast, only one out of five advanced plaque endothelial samples stained positively for CD81, only in the shoulder region of the plaque, corresponding to the newly developing site of the lesion (Supplementary material online, Figure S1B). The CD81 staining was endothelial, as is best seen in the higher magnification of Figure 1B, and from additional staining for the endothelial marker CD31 and macrophages (Figures 1A and Supplementary material online, Figure S2). In addition, anti-CD81 antibodies also showed some intimal staining in the plaque areas, probably representing infiltrating macrophages, as monocyte/macrophages also express CD81. In summary, both RNA and protein analyses indicate that CD81 is upregulated in the endothelial cells covering human early atherosclerotic lesions.


Figure 1
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  		Figure 1 CD81 is specifically upregulated in the endothelium of early human atherosclerotic lesions. (A) Immunohistochemical analysis of CD81 expression in early atherosclerosis in donors 12 and 14 (upper panels). The early plaque is shown in the right panels (red), whereas the healthy vessel wall of the proximal artery is in the left panels (black). CD81 immunopositivity in endothelial cells is indicated by arrows. Endothelium-specific CD31 staining is shown in middle panels. The lower panels and the overview slides are stained by the macrophage-specific Ham56 dye. Immunohistochemical slides from the remaining donors are shown in Supplementary material online, Figure S1. (B) Detailed view of CD81 staining over the early atherosclerotic plaque in donor 12. Donor codes and information can be found in ‘Methods’ and in Volger et al.8 Scale bars represent 100 µm, unless stated otherwise.

 
3.2 CD81 is upregulated by oxidative stress
To determine the conditions responsible for CD81 upregulation, confluent monolayers of HUVEC were exposed to various stimuli in vitro as described in ‘Methods’ and in the legend to Figure 2A. Data presented in this figure show that CD81 mRNA expression was largely unaffected by standard pro-inflammatory and pro-atherogenic stimuli, such as TNF{alpha}, IL-1β, histamine, or TGFβ, by a combination thereof, or by mechanical stimulation by stretch. In contrast, HUVEC monolayers cultured under shear stress significantly increased CD81 mRNA expression. Because shear stress effectively increases oxidative stress in endothelial cells,24 HUVEC monolayers were also treated with oxidative stress inducers tert-butyl hydrogen peroxide (tb-H2O2) and PMS, both of which significantly increased CD81 mRNA. Similarly, exposure of HUVEC monolayers to macrophage-conditioned medium in the presence of oxidated LDL also resulted in somewhat increased mRNA expression, but this did not reach significance. Furthermore, the exposure of HUVEC to PMS or shear stress also increased CD81 expression at the protein level, as determined by immunofluoresecent staining (Figure 2B). These results indicate that CD81 upregulation is driven by oxidative stress, rather than by inflammation.


Figure 2
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  		Figure 2 (A) Oxidative stress upregulates CD81 mRNA. HUVEC monolayers were exposed to various inflammation-related stimuli and to stretch for 24 h, to laminar flow for 6 days, or to oxidative stress-related stimuli for 16 h. The cells were harvested, total RNA isolated, and CD81 mRNA was quantified by RT–PCR. Ang1, Angiopoietin 1; Hist, histamine; OxLDL MM, medium conditioned by OxLDL-stimulated macrophages. (B) Upregulation of CD81 protein. HUVEC monolayers were exposed to PMS for 20 h or to laminar flow for 24 h, the cells were fixed and immunostained with anti-CD81 antibody. (C) Up- and downregulation of CD81 mRNA by lentiviral vectors. HUVEC monolayers were transduced with lentiviral particles carrying constructs containing cDNA of CD81, CD81-directed shRNA cassette, or control particles lacking the CD81 cDNA (mock) or containing a firefly luciferase-specific shRNA cassette (control si). Cells were harvested after 4 days, total RNA was isolated, and CD81 mRNA was quantified by RT–PCR. (D) The same as in (C), only the cells were released from the growth matrix by trypsin, stained with anti-CD81 antibody, and assayed by flow cytometry. Filled histogram, negative control (secondary antibody only on mock-transduced cells); thin dashed line, CD81 shRNA-transduced cells; thick dashed line, firefly luciferase shRNA-transduced cells; thick line, mock-transduced cells; thin line, CD81-transduced cells. Data represent the mean (±SEM) of experiments from three independent HUVEC isolates (A and C), or typical experiments (B and D). **P < 0.01, *P < 0.05.

 
3.3 CD81 overexpression and silencing
In order to investigate the consequences of elevated CD81, we overexpressed human CD81 from lentiviral vectors in a HUVEC model system, which was previously extensively validated in vivo.7,25 In parallel, we also introduced a lentivirally expressed, CD81-directed shRNA in order to explore the opposite scenario, CD81 downregulation. These procedures resulted in efficient up- or downregulation of CD81 at both mRNA and protein level, as determined by semi-quantitative RT–PCR and flow cytometry analysis (Figure 2C and D), but did not lead to any obvious alteration of the endothelial phenotype (see Supplementary material online, Figure S5).

3.4 CD81 overexpression is not associated with increased monocyte rolling
Upregulation of CD81 in early atherosclerosis suggests that it could participate in the recruitment of inflammatory cells to the lesion site. We therefore tested the effect of CD81 on monocyte rolling, an early step in the interaction with endothelial cells.5 To this end, lentivirally transduced HUVEC on glass coverslips were stimulated or not with TNF{alpha} for 16 h, placed into the perfusion apparatus and the primary monocytes were passed over the monolayer. The experiments were followed in real time, and the number of rolling monocytes for individual conditions was scored by analysing the video recordings. As shown in Figure 3, neither upregulation nor downregulation of CD81 expression affected the rate of monocyte rolling over both the unstimulated and stimulated HUVEC monolayers.


Figure 3
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  		Figure 3 CD81 overexpression in the endothelium does not increase monocyte rolling. Lentivirally transduced HUVEC were grown on glass coverslips till confluency, stimulated (B) or not (A) with 10 ng/mL TNF{alpha} for 16 h, and the percentage of rolling primary monocytes was measured from video recordings as indicated in ‘Methods’. Data represent the mean of six independent measurements on two HUVEC isolates (±SEM).

 
3.5 CD81 overexpression enhances monocyte adhesion to endothelium under flow
Considering the upregulation of CD81 in the endothelium of early atherosclerotic plaques, we were particularly interested whether the increased endothelial CD81 could stimulate firm monocyte adhesion in the absence of a full-blown inflammatory response. Lentivirally transduced HUVEC were treated as described above, and the amount of firmly bound monocytes was scored. Interestingly, CD81 overexpression resulted in a significantly increased number of monocytes firmly adhering to the unstimulated HUVEC monolayers (Figure 4A), whereas no effect was observed when CD81 expression was silenced by shRNA. Next, a similar experiment was performed, this time after a 16 h TNF{alpha} stimulation of the HUVEC monolayers (Figure 4B). As expected, the basal adhesion of monocytes was increased by TNF{alpha}, when compared with unstimulated, mock-transduced HUVEC. Surprisingly, the overall level of monocyte adhesion to non-activated CD81-overexpressing HUVEC was very similar to the numbers attained in TNF{alpha}-stimulated, mock-transduced HUVEC (Figure 4A and B). CD81 overexpression in TNF{alpha}-stimulated HUVEC was associated with only a slight but significant further increase of monocyte adhesion when compared with mock-transduced HUVEC. As before, the CD81 knock down did not result in a significant change in monocyte adhesion. Given the upregulation of CD81 expression by oxidative stress, monocyte adhesion was also investigated for PMS-treated HUVEC monolayers (Figure 4C). Similarly to CD81 overexpression, PMS treatment resulted in a significantly increased monocyte adhesion. In conclusion, monocyte adhesion to endothelial cells is significantly increased by induced expression of CD81, especially in the absence of TNF{alpha}-stimulation, but is not significantly altered by downregulation of basal CD81 levels.


Figure 4
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  		Figure 4 Endothelial CD81 upregulation enhances monocyte adhesion. (A and B) Lentivirally transduced HUVEC were grown on gelatin/fibronectin-coated glass coverslips till confluency, stimulated (B) or not (A) with 10 ng/mL TNF{alpha} for 16 h. (C) HUVEC were stimulated for 24 h with 10 µM PMS. The adhesion of primary monocyte under flow conditions was measured as indicated in ‘Methods’. Data represent the mean of nine independent measurements on three HUVEC isolates (±SEM), *P < 0.05, **P < 0.01.

 
3.6 CD81 upregulation in endothelial cells is not associated with increased surface expression of ICAM-1 and VCAM-1
The possibility exists that CD81, similarly to related tetraspanins CD9 and CD151, could increase the surface expression of endothelial adhesion molecules ICAM-1 and VCAM-1 that mediate firm adhesion of monocytes to the endothelium.5,13 To test this, confluent CD81- and mock-transduced HUVEC were stimulated or not with TNF{alpha} for 16 h, resuspended by trypsin, immunostained for surface exposed VCAM-1 and ICAM-1, and analysed by flow cytometry. As expected, the endothelial expression of VCAM-1 and ICAM-1 was enhanced upon TNF{alpha} stimulation by about two orders of magnitude (Figure 5A). In contrast, little or no increase in expression of both adhesion molecules was observed upon CD81 overexpression, in both TNF{alpha}-stimulated and unstimulated HUVEC. Thus, CD81 overexpression is unlikely to stimulate monocyte adhesion by increasing the total surface expression of endothelial adhesion molecules such as ICAM-1 and VCAM-1.


Figure 5
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  		Figure 5 CD81 upregulation in endothelial cells does not substantially increase ICAM-1 or VCAM-1, but CD81-mediated enhancement of monocyte adhesion is VCAM-1 and ICAM-1-dependent. (A) Lentivirally transduced HUVEC were grown to confluency, stimulated or not by 10 ng/mL TNF{alpha} for 16 h, released from the matrix by typsin and stained for indicated proteins as described in ‘Methods’. Thin line, secondary antibody only on mock-transduced cells; thin line with the filled histogram, mock-transduced cells; thick line, CD81 transduced cells. (B) Lentivirally transduced HUVEC was grown on gelatin/fibronectin-coated glass coverslips till confluency, incubated with indicated antibodies for 30 min, followed by measurement of monocyte adhesion under flow as described in ‘Methods’. Data represent the mean of eight independent measurements on four HUVEC isolates (±SEM), *P < 0.05.

 
3.7 The CD81-mediated enhancement of monocyte adhesion to the endothelium is ICAM-1- and VCAM-1-dependent
Even though CD81 does not increase endothelial expression of ICAM-1 and VCAM-1, the CD81-mediated enhancement of monocyte adhesion may still be dependent on those two adhesion molecules. To clarify the issue, we performed the monocyte adhesion assay under flow conditions in the presence of ICAM-1 and VCAM-1-directed antibodies (Figure 5B). After a 30 min pre-incubation of HUVEC monolayers with a control, non-blocking antibody, CD81 overexpression in HUVEC was still associated with significantly increased monocyte adhesion when compared with mock-treated cells. The difference in monocyte adhesion between CD81- and mock-transduced HUVEC cells was also present when these monolayers were pre-treated with anti-ICAM-1 or anti-VCAM-1 antibodies separately. When the two antibodies were combined, however, the enhancing effect of CD81 overexpression disappeared almost completely. These results show that the CD81-mediated enhancement of monocyte adhesion is ICAM-1 and VCAM-1 dependent.

3.8 Endothelial CD81 colocalizes with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes
Given the stimulating effect of CD81 on monocyte binding by ICAM-1 and VCAM-1, one might expect that CD81 would be enriched in the areas of endothelial cell membrane that are in direct contact with a bound monocyte, and that CD81 should thus also colocalize there with ICAM-1 and VCAM-1. To test this hypothesis, HUVEC were transduced with a lentiviral V5-tagged CD81 construct, treated with TNF{alpha} for 16 h on glass coverslips, monocytes were added for 30 min, the cultures were fixed, and V5-tagged CD81 was detected by anti-V5 antibody. This allowed specific detection of endothelial CD81, because monocytes also express this surface marker, and that would otherwise also be detected if a CD81-directed antibody was used. As shown in Supplementary material online, Figure S3, the V5 antibody indeed specifically detected endothelial CD81-positive adhesion rings around the bound monocytes, visualized by F-actin staining. Next a triple labelling was performed with anti-V5 antibody, anti-ICAM-1 antibody, and Phalloidin (to visualize F-actin). The merged panel (Figure 6A) shows that CD81 and ICAM-1 colocalize in the ring structures, and that endothelial CD81, together with ICAM-1, may be part of the transmigratory cups detected at the sites of cell adhesion,26 as indicated in the X–Z projection of the confocal stack (Supplementary material online, Figure S4A). Virtually identical results were obtained for VCAM-1 (Figure 6A and Supplementary material online, Figure S4A). Unfortunately, this experiment could not be performed in the absence of TNF-stimulation, as the ICAM-1 and VCAM-1 antibodies did not give sufficient signal in the non-stimulated cells for proper visualization. Still, these data demonstrate that endothelial CD81 is present at the contact sites between endothelial cells and monocytes, it colocalizes there with the archetypal endothelial adhesion molecules ICAM-1 and VCAM-1, and participates in the formation of transmigratory cups.


Figure 6
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  		Figure 6 Endothelial CD81 colocalizes with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes and enhances ICAM-1 and VCAM-1 clustering. (A) HUVEC were lentivirally transduced with a V5-tagged CD81 construct, grown on gelatin/fibronectin-coated glass coverslips to confluency, and primary monocytes were allowed to adhere to the monolayer for 30 min. The cells were then fixed and immunostained for the V5 tag and indicated proteins. (B and C) HUVEC were transduced with mock- (upper panels) or CD81- (lower panels) encoding lentiviruses, grown to confluency on gelatin/fibronectin-coated glass coverslips, fixed and stained for ICAM-1 (B), VCAM-1 (C) and VE-cadherin. Enhanced ICAM-1 and VCAM-1 clustering in CD81 overexpressing cells is indicated by white arrowheads, asterisk in (C) denotes the cell without CD81 overexpression. GFP identifies lentivirally transduced cells. (D) The same as in (B), but the monolayers were grown on thermanox, pre-embedment labelling for CD81 (5 nm gold, open arrowheads) and ICAM-1 (10 nm gold, closed arrowheads) was performed, thin sections were cut and analysed by electron microscopy as described in ‘Methods’. The left panel depicts mock-transduced cells, whereas the right panel CD81-transduced cells.

 
3.9 Overexpression of endothelial CD81 results in altered cellular localization and enhances clustering of ICAM-1 and VCAM-1
As described earlier, the stimulation of monocyte adhesion by CD81 overexpression is not mediated by increased total ICAM-1 or VCAM-1 surface expression, yet it is ICAM-1 and VCAM-1 dependent. We, therefore, considered the possibility that CD81 could modulate the localization of ICAM-1 and VCAM-1 on the endothelial cell membrane and thereby increase the adhesion potency of monocytes. To address this issue, HUVEC cells were transduced with CD81 and mock lentiviral constructs, prepared for immunochemistry, and immunostained for ICAM-1 or VCAM-1 molecules. In the mock-transduced cells, the ICAM-1 and VCAM-1 staining patterns were consistent with a rather diffuse membrane distribution of the adhesion molecules with somewhat higher concentration along cell junctions, as visualized by staining for junction-specific VE-cadherin (Figure 6B and C). In contrast, CD81 overexpression resulted in distinct relocalization of ICAM-1 molecules into larger membrane clusters that were still centred in junction areas, but extended further along the cell membrane (Figure 6B). Similarly, VCAM-1 staining upon CD81 overexpression became distinctly cluster-like, with clearly higher concentration of VCAM-1 in the cell junction areas, as indicated by increased colocalization with VE-cadherin between cells overexpressing CD81 (indicated by arrowheads in Figure 6C). Compared to the overexpression, CD81 knockdown was associated with little, if any, changes in ICAM-1 and VCAM-1 staining patterns (see Supplementary material online, Figure S4B). Next, the mock- and CD81-transduced HUVEC were investigated in more detail by electron microscopy. The non-stimulated cells were fixed, double labelled for CD81 and ICAM-1, and the distribution of ICAM-1 and CD81 molecules was examined. As shown in Figure 6D, in mock-transduced endothelial cells ICAM-1 and CD81 both exhibited a typical membrane staining which was not very strong. This staining became more prominent and assumed a distinct patchy appearance in the cells overexpressing CD81, with a high degree of colocalization of CD81 molecules in the clusters of ICAM-1 molecules. These results suggest that increased endothelial CD81 expression results in a redistribution of ICAM-1 and VCAM-1 within the endothelial membrane, and formation of membrane clusters containing ICAM-1, VCAM-1, and CD81 molecules.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 Supplementary material
 References
 
Atherosclerosis is characterized by its focal nature and localization of lesions in the areas of disturbed blood flow along the vascular tree. These sites are inflammation-prone,27 and during plaque formation display increased expression of inflammation-sensitive endothelial adhesion molecules VCAM-1 and ICAM-1 of the immunoglobulin superfamily.28–31 In the current study, we present evidence that tetraspanin family member CD81 is also upregulated in atherosclerotic lesions, particularly in the endothelium overlaying the early human atherosclerotic plaques (Figure 1 and Supplementary material online, Figure S1).8 Surprisingly, CD81 positivity in advanced human atherosclerotic lesions was much diminished when compared with the early lesions, corresponding well with our earlier transcriptome analysis.8 The presence of CD81 especially in early lesions suggests that CD81 could play a role in the initial stages of lesion formation, a role that diminishes when the lesion matures, acquires a fibrous cap and stabilizes.16 In marked contrast to CD81, we did not see any upregulation of related tetraspannins CD9 and CD151 in atherosclerotic lesions,8 which further supports the specific role of CD81 in endothelium during early atherosclerosis.

In contrast to ICAM-1 and VCAM-1, CD81 expression seems to be induced by oxidant stress, independent of inflammation. Whereas various pro-inflamatory and atherogenic stimuli, combinations thereof, or exposure to stretch had little effect on CD81 expression levels, oxidative stress-related stimuli consistently resulted in CD81 upregulation on the mRNA- as well as protein level (Figure 2). Surprisingly perhaps, CD81 expression was increased by laminar flow, even though it is usually considered atheroprotective. Laminar flow, however, does increase oxidative load in endothelial cells, which is normally counteracted by specific protective mechanisms,24 and also induces ICAM-1.32 It is likely that the endothelium at the lesion sites is exposed to even higher levels of oxidative stress and possibly other factors, which ensure upregulation of CD81 above the level maintained in the healthy vessel wall, giving it an expression pattern similar to ICAM-1.8 Analysis of CD81 promoter as described7,24 did not identify any obvious oxidative stress-related transcription factor binding sites or the antioxidant response element (ARE); therefore, the precise mechanism of CD81 upregulation remains unknown at this time.

The role elevated endothelial CD81 may play in early atherosclerosis is enhanced monocyte adhesion (Figure 4), which further involves endothelial ICAM-1 and VCAM-1 adhesion molecules.5 These are normally present on the non-activated endothelium at low, but detectable levels,7 although their surface expression is dramatically increased by inflammation (Figure 5).33 In our experiments, we found that increased CD81 expression in non-activated endothelial cells increased monocyte adhesion nearly to the level of TNF{alpha}-activated endothelium (Figure 4), indicating that endothelial cells with high CD81 do not require cytokine-invoked inflammatory stimulation in order to efficiently capture monocytes. Furthermore, the enhancement of monocyte adhesion upon oxidative stress treatment of the monolayers was similar to that invoked by CD81. As the stimulatory effect of CD81 is blocked by the addition of a mixture of anti-ICAM-1 and VCAM-1 antibodies (Figure 5B), it appears that CD81 is an accessory factor that requires both adhesion molecules in order to increase monocyte adhesion. This is further supported by the observation that CD81 colocalizes with ICAM-1 and VCAM-1 in adhesion rings formed by the endothelial membrane around the bound leukocytes (Figure 6A and Supplementary material online, Figure S4A), clearly showing a potential for CD81/ICAM-1/VCAM-1 interactions where it matters.26

In contrast to related tetraspanins CD9 and CD151 that promote surface expression of ICAM-1 and VCAM-1 on endothelial cells,13 CD81 overexpression led to only a negligible increase of ICAM-1 and VCAM-1 surface expression (Figure 5A). Rather, confocal and electron microscopy support the notion that available ICAM-1 and VCAM-1 molecules are clustered and relocalized by CD81 presence (Figure 6), increasing the local concentration particularly around the cell junctions, where CD81 concentration is the highest.15 Interestingly, in CD81 overexpressing cells, ICAM-1 staining patterns, with clusters in the plasma membrane extending from the junction areas, are similar to the staining patterns in the atherosclerosis-prone regions of the vascular tree.30 VCAM-1 staining was similar to ICAM-1, but the clusters induced seemed to be somewhat smaller and VCAM-1 was more concentrated in junction areas (Figure 6C). It seems that CD81, similarly to other tetraspanins,11 may form raft-like membrane structures that increase local ICAM-1 and VCAM-1 concentration and explain the higher efficiency of monocyte adhesion to CD81 expressing cells. In this way, even the relatively low concentrations of ICAM-1 and VCAM-1 on the non-activated endothelial cells could cooperatively provide sufficiently strong binding sites to efficiently arrest the rolling monocytes. This is also consistent with the relatively larger effect we observed upon CD81 overexpression in non-activated endothelial cells when compared to TNF{alpha}-activated cells, where the concentration of ICAM-1 and VCAM-1 is already sufficient to account for efficient monocyte adhesion. It may seem surprising, perhaps, that the downregulation of basal endothelial CD81 by specific shRNA did not result in any discernable functional effects. This is in contrast with results reported by Barreiro et al.13 for two related tetraspanins CD9 and CD151, which act through increasing surface expression of VCAM-1 and ICAM-1, or with results of Feigelson et al.14 for CD81 interaction with integrins in monocytes. Under normal circumstances in healthy endothelium, CD81 apparently contributes only little to monocyte adhesion, as suggested by the minimal changes induced in ICAM-1 or VCAM-1 staining patterns upon CD81 knockdown by shRNA (Supplementary material online, Figure S4B). With increased CD81 expression, however, its contribution to monocyte binding could become apparent and detectable in functional assays, as only high enough CD81 levels would induce the observed clustering effect.

In conclusion, the present study has identified CD81 as a likely candidate to be one of the major factors in the initiation of atherosclerotic lesions, as it combines an ability to increase monocyte adhesion to non-inflamed endothelium with a nearly perfect specific upregulation in the endothelium of early atherosclerotic lesions. As such, it would be able to initiate the infiltration of monocytes into the vessel wall, preceding the full-blown inflammatory reaction of the vessel wall by macrophage-derived cytokines that are necessary to induce high expression levels of endothelial adhesion molecules.


   Funding
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 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
Supplementary material
 References
 
SenterNovem, The Hague, The Netherlands (grant IGE03012C); Netherlands Organization for Scientific Research (grant 050-110-1014); European Union (grant LSHM-CT-2003-1503254). P.L.H. is a fellow of the Landsteiner Foundation for Blood Transfusion Research (project no. 203). J.D.v.B. is supported by the Netherlands Heart Foundation (grant no. 2005T039).


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 Supplementary material
References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Notes
 
{dagger} Present address. Institute of Biotechnology, Czech Academy of Sciences, Prague, Czech Republic. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 Supplementary material
 References
 

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