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Cardiovascular Research 2001 50(3):547-555; doi:10.1016/S0008-6363(01)00207-3
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Copyright © 2001, European Society of Cardiology

Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells: induction of VCAM-1 but no proliferation

Charlotte Lawson, Mark E Ainsworth, Ann M McCormack, Magdi Yacoub and Marlene L Rose*

Transplant Immunology Group, Imperial College School of Medicine, National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Harefield, Middlesex UB9 6JH, United Kingdom

* Corresponding author. Tel.: +44-1895-828-575; fax: +44-1895-828-900 marlene.rose{at}harefield.nthames.nhs.uk

Received 20 November 2000; accepted 8 January 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Intercellular adhesion molecule (ICAM)-1 is an immunoglobulin-like cell adhesion molecule expressed by several cell types, including proliferating vascular smooth muscle cells (VSMC). Cross-linking ICAM-1 on the surface of different cell types has previously been shown to cause an increase in cellular activation within the cytoplasm. Here, our objective was to examine events following ligation of ICAM-1 on the surface of human VSMC. Methods: VSMC were isolated by explant from human pulmonary arteries or aortic tissue from cardiac transplant donors. ICAM-1 was ligated with monoclonal antibodies, followed by cross-linking with a secondary antibody. Activation of signalling pathways, proliferation and expression of a second adhesion molecule, vascular cell adhesion molecule (VCAM)-1 were investigated. Results: ICAM-1 cross-linking caused an increase in activation of extracellular regulated kinase (Erk)-1/-2 and Jun N-terminal kinase (JNK)-1/-2. mRNA and protein for VCAM-1 was observed after ICAM-1 cross-linking, and this was abrogated by addition of an upstream inhibitor of Erk-1/-2, PD98059. No increase in cell proliferation was observed. Conclusions: Ligation of ICAM-1 on the surface of vascular smooth muscle cells in vitro, leads to the expression of adhesion molecules associated with monocyte infiltration, but does not contribute to smooth muscle cell proliferation. In vivo, this might lead to prolongation of the inflammatory response within diseased blood vessels, by arresting monocytes within atherosclerotic plaques.

KEYWORDS Erk, extracellular regulated kinase; FGF, fibroblast growth factor; HUVEC, human umbilical vein endothelial cells; ICAM, intercellular adhesion molecule; JNK, Jun N-terminal kinase; LFA, leukocyte function-associated antigen; MAPK, mitogen activated protein kinase; MEK, MAPK/Erk kinase; MHC, major histocompatibility complex; PAGE, polyacrylamide gel electrophoresis; PDGF, platelet derived growth factor; PSI, proteasome inhibitor I; RAM, rabbit anti-mouse Ig; TCA, trichloroacetic acid; TGF, transforming growth factor; VCAM, vascular cell adhesion molecule; VLA, very late antigen; VSMC, vascular smooth muscle cells


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Atherosclerosis is the principal cause of death in Western civilisation. The atherosclerotic lesion is characterised by the accumulation of intimal vascular smooth muscle cells (VSMC) together with variable numbers of macrophages and T lymphocytes, the presence of lipids, particularly in the form of cholesteryl esters, and the formation of large amounts of connective tissue matrix [1–3]. The presence of VSMC is also a feature of transplant atherosclerosis and restenosis injury [4]. VSMC, which migrate from the media of the artery wall to the intima have been extensively characterised both from human lesions and from animal models of atherosclerosis. These cells acquire a synthetic phenotype, i.e. they lose contractile functions but express various matrix proteins, particularly proteoglycans, collagen and elastic fibres as well as growth factors including PDGF and FGF, both of which can cause smooth muscle cell proliferation [5–9]. They also express intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, two adhesion molecules involved in leukocyte trafficking across endothelial cells and possibly in VSMC migration [10–17].

ICAM-1 (CD54) is a 90-kDa member of the Ig superfamily and is also found on the surface of several other cell types, including leukocytes and endothelial cells. It has been shown to have a number of different ligands including LFA-1, Mac-1, fibrinogen, rhinoviruses and plasmodium falciparum [18]. The role of ICAM-1 as an adhesive molecule has been described in detail elsewhere [18]. It is now clear, however, that ICAM-1 is also able to transmit signals after ligation at the cell surface [19]. Using cross-linked antibodies to mimic its interaction with its ligands, ligation of ICAM-1 has been shown to induce tyrosine phosphorylation of cortactin [20], and activation of the Rho signalling pathway [21,22] in rat brain endothelial cells. Cross-linking ICAM-1 on the surface of human umbilical vein endothelial cells (HUVEC) results in expression of VCAM-1, a process which is independent of NF-{kappa}B activity [23]. Tyrosine phosphorylation of the cell cycle protein, cdc2 kinase, occurs in T cells [24], whilst peripheral blood mononuclear cells [25]) and synovial cells [26] are also activated by ICAM-1 cross-linking. ICAM-1 signalling in VSMC has not been investigated. Signal transduction via ICAM-1 could have implications for the pathogenesis of vascular diseases where VSMC migrate to the vessel intima. For example, interaction between ICAM-1 on VSMC and LFA-1 on infiltrating leukocytes could help maintain the chronicity of the lesions.

Here, we have cross-linked ICAM-1 on the surface of human vascular smooth muscle cells in order to determine the down stream signalling events and possible biological end-points of such signalling.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
VSMC were isolated from pulmonary arteries or aortic tissue from donors at heart transplant, by explant. The collection of this tissue was approved by the local Ethics Committee. Briefly, tissue was collected in HBSS medium (Gibco L.T.I., Paisley, UK). After removal of the adventitia and surrounding connective tissue, the remaining intimal tissue was pinned onto tissue culture plastic (Helena Biosciences, Sunderland, UK) using sterile 21-gauge needles, and was bathed in full VSMC medium: DMEM containing 1000 mg/l glucose (low glucose DMEM) supplemented with 2 mM L-glutamine, 150 u/ml penicillin/streptomycin, 25 mM Hepes pH 7.5, 1xnon-essential amino acid solution and 15% FCS (all from Sigma, Poole, UK). Explant cultures were maintained at 37°C in a 5% CO2 atmosphere. VSMC were allowed to migrate out onto the plastic over a period of 2–8 weeks. Once there were large colonies of VSMC adhered to the plastic these were trypsinised and maintained as monolayers in full VSMC medium, as above. Confluent monolayers of single isolates at between passages 3–10 were used for all experiments. Cells were identified as VSMC by positive staining for smooth muscle {alpha}-actin, a recognised marker of smooth muscle cells [27] (see below). Results are shown for individual isolates derived from aorta or pulmonary artery. We have seen no differences between VSMC from these two sources.

2.2 Antibodies
Protein-G purified fractions of anti-ICAM-1 clone 6.5B5 (isotype IgG1, a kind gift of Professor D. Haskard; National Heart and Lung Institute, London, UK; [28] were used at 15 µg/ml. Rabbit anti-mouse Ig Ab Z0259 (RAM; Dako, Cambridge, UK) was used at 1:100 for cross-linking. Protein G purified fractions of CRL1724 (ATCC), an IgG1 isotype MAb that recognises v-myb and chicken c-myb, were used at 20 µg/ml as an irrelevant antibody control. Protein G purified fractions of anti-VCAM-1 clone 1.4C3 (a kind gift of Professor D. Haskard; National Heart and Lung Institute, London, UK; [28] were used at 250 ng/ml for blotting and 2.5 µg/ml for immunoprecipitation. Anti-VCAM-1 mAb BBIG-VI (R+D Systems, Abingdon, UK) was used at 10 µg/ml for blotting and immunoprecipitation. Anti-active Erk-1 (Promega, Southampton UK) was used at 1:5000 for blotting. Anti-Erk-1 (Transduction Laboratories; purchased from Affiniti Biosystems, Exeter UK), anti-active JNK-1 and anti-JNK-1 (Santa Cruz, purchased from Insight Biotechnology, London, UK) were used at 1:400 for blotting. Anti-I{kappa}B{alpha} (Santa Cruz) was used at 1:200 for blotting. Goat anti-murine and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson (Stratech, Luton, UK) and were used at 1:5000 as secondary antibodies. Protein-G purified fractions of anti-human MHC class I (clone W6/32 ATCC) were used at 10 mg/ml for indirect immunofluorescence staining. Anti-smooth muscle {alpha}-actin (Clone 1A4; Sigma) was used at 1:100 for indirect immunofluorescence staining or 1:1000 for blotting. Rabbit anti-mouse Ig F(Ab)2-FITC (F0313, DAKO, Cambridgeshire, UK) was used at 1:30 as a secondary antibody for flow cytometry or immunocytochemistry. Goat anti-mouse Ig F(Ab)2-ALEXA 594 nm (Molecular Probes, Leiden, Netherlands) was used at 1:250 for immunocytochemistry.

2.3 Determination of SMC phenotype
Confluent monolayers of VSMC were trypsinised and stained for the presence of surface ICAM-1 and MHC class I, followed by goat anti-mouse Ig F(Ab)2-FITC. Cells were analysed using a Coulter EPICS flow cytometer. Alternatively, 5x104 HVSMC were plated onto sterile coverslips and allowed to adhere for 48 h. Coverslips were washed in PBS and then fixed for 5 min in ice-cold acetone. They were stained for the presence of smooth muscle {alpha}-actin, ICAM-1 or MHC class I, followed by goat anti-mouse Ig F(Ab)2-ALEXA 594 nm. Positively stained cells were visualised with a fluorescent microscope (Axiophot, Zeiss, Hertfordshire, UK). VSMC selected for experiments expressed both smooth muscle {alpha}-actin and ICAM-1.

2.4 Western blotting and immunodetection
VSMC were plated onto 6-cm dishes. Once confluent, the cells were washed in serum-free DMEM and then incubated in DMEM with 1% FCS for 18 h in order to minimise activation by serum. Cells were incubated in DMEM; 1% FCS for the duration of experiments. Anti-ICAM-1 antibody was added and the cells were incubated for 30 min at 37°C. Cells were washed once with serum free DMEM and incubated with RAM at a dilution of 1:100. As a positive control, VSMC were incubated with 100 u/ml recombinant human TNF (R+D Systems) together with 100 u/ml IL-1β (Insight Biotechnology) for 15 min at 37°C. Monolayers were harvested as described previously [23]. Protein (25 µg) was electrophoresed on 10% polyacrylamide gels and blotted onto nitrocellulose membrane (Amersham International; Amersham, UK). After blocking in 5% non-fat milk; PBS; 0.01% Tween-20 (marvel PBS-T), membranes were incubated in primary antibodies for 1 h with agitation followed by three washes in PBS-T. The membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase and after three washes in PBS-T, incubated in ECL reagents (Amersham) and exposed to autoradiography film (Amersham).

2.5 Proliferation assays
VSMC were plated onto 24-well dishes at 2.5x104 cells per well. Cells were quiesced in DMEM with 1% FCS for 48 h. They were washed in serum-free DMEM and replaced with 0.5 ml DMEM with 1% FCS. They were then incubated with anti-ICAM-1 antibody with or without RAM, at 37°C for up to 72 h. As a positive control, VSMC were incubated with 100 u/ml recombinant human PDGF-BB (R+D systems) for 48 h at 37°C. [3H] TdR was added to all wells for the last 24 h of culture. The medium was removed and 0.5 ml ice cold 10% trichloroacetic acid (TCA) was added to each well. Cells were incubated for 20 min at 4°C, washed once with 1 ml distilled water, and then lysed in 0.1 M SDS; 0.3 M NaOH at 37°C for 1.5 h with agitation. Lysates were removed from the wells and placed into scintillation vials containing 3 ml scintillation fluid (Packard, Pangbourne, UK). Vials were counted for 1 min on a Beta counter (1600TR, Packard).

2.6 Immunoprecipitation
Cells were stimulated and lysed as described above for Western blotting in Section 2.4. Each cell lysate (100 µg) was incubated with primary antibody and 20 µl protein-G sepharose slurry (Sigma) for 18 h at 4°C with mixing. Beads were pelleted and washed four times in RIPA buffer, before being resuspended in 100 µl SDS gel loading buffer without reducing agents. The beads were boiled for 5 min and pelleted again. Supernatants were electrophoresed on 10% polyacrylamide gels, blotted and probed as described above.

Cells were pretreated with proteasome inhibitor I (PSI; Calbiochem, Nottingham, UK) at a final concentration of 10 µM in order to inhibit NF-{kappa}B activation [29], or with 30 µM PD98059 (Calbiochem) to inhibit Erk activity [30,31].

2.7 RT-PCR
VSMC were plated onto 24-well dishes. Confluent monolayers were allowed to quiesce in DMEM with 1% FCS for 18 h. Cells were washed in serum-free DMEM and replaced with 0.5 ml DMEM with 1% FCS. Anti-ICAM-1 antibody was added with or without RAM and cells were incubated at 37°C for up to 72 h. The medium was removed from each well and snap frozen on dry ice followed by storage at –70°C for further analysis. Total RNA was extracted from the cells using Purescript total RNA isolation kits (Flowgen, Staffordshire, UK) or TRI Reagent (Helena Biosciences; Sunderland, UK) according to the manufacturer's instructions. RT-PCR was carried out using a one-step protocol. Briefly, cDNA synthesis and PCR was carried out using 5 units AMV reverse transcriptase (Promega), 7 units TFL polymerase (Promega) and 25 nmol specific oligodeoxynucleotide primer pairs for β-actin or VCAM-1 (MWG Biotech, Germany; for sequences see Ref. [23]) together with the manufacturers buffer, 200 µmol dNTP and 12.5 nmol MgSO4. Two microlitres of each RNA sample was used per RT-PCR reaction. PCR was performed in a Hybaid Omnigene thermal cycler (Hybaid, Middlesex, UK) with an initial incubation of 48°C for 45 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, followed by a final incubation at 68°C for 7 min. VCAM-1 primers were designed to span intron-exon boundaries. The upstream primer bound between bp1424–1442 (exon VII, Ig domain 6) and the downstream primer bound between 1683 and 1664 (exon VIII, Ig domain 7) of human VCAM-1 cDNA [32], with an expected product of 260 base pairs [33]).

2.8 Statistics
Mann–Whitney U nonparametric tests were performed using Sigma-Stat (SPSS, San Rafael, CA), for all statistics presented here.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Expression of ICAM-1 by cultured VSMC
Cultured aortic and pulmonary artery VSMC used in this study were positive for cell surface expression of ICAM-1 and MHC class I, analysed by flow cytometry (Fig. 1) and indirect immunofluorescence (not shown). The VSMC used also expressed smooth muscle {alpha}-actin (data not shown).


Figure 1
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  		Fig. 1 ICAM-1 expression of cultured VSMC. Confluent monolayers of VSMC derived from human aorta were trypsinised and stained with anti-ICAM-1, anti-HLA class I or an irrelevant isotype control antibody, followed by RAM-FITC. Cells were analysed by flow cytometry using a Coulter EPICS analyser (Beckman Coulter, UK.). Solid line; control stained cells, Dashed line; MHC class I stained cells; Dotted line; ICAM-1 stained cells. Representative of all isolates used in this study.

 
3.2 Activation of signalling cascades after ICAM-1 cross-linking on the surface of VSMC
In agreement with our previous results after ICAM-1 cross-linking on the surface of HUVEC [23], activation of extracellular regulated kinase (Erk)-1/-2 was observed within 5 min of cross-linking VSMC with anti-ICAM-1 antibodies (Fig. 2A). This returned to basal levels of activity by 30 min after cross-linking. Ligation of ICAM-1 for 10 min, without cross-linking also induced Erk-1/-2 activity. There was no effect of incubation of cells for 10 min with CRL1724, an isotype matched control antibody. Incubation of VSMC with TNF/IL-1β for 15 min also induced high levels of Erk-1/-2 activation, as expected (Fig. 2A). Pre-incubation of VSMC with PD98059, an inhibitor of the upstream kinase MAPK/Erk kinase ((MEK); [30,31]), abrogated the Erk-1/-2 activity seen (Fig. 2A).


Figure 2
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  		Fig. 2 Activation of MAPK-like pathways after ICAM-1 cross-linking of VSMC. Resting VSMC from human aorta were treated with 15 µg/ml anti-ICAM-1 MAb for 30 min at 37°C, followed by RAM for the indicated times, ±pre-treatment with 30 µM PD98059. As negative controls they were treated with irrelevant antibody alone, anti-ICAM-1 MAb alone, RAM alone for 10 min. Cells were treated with 100 u/ml TNF and 100 u/ml IL-1β for 15 min as a positive control. Cells were lysed, resolved by SDS–PAGE and blotted onto nitrocellulose as described in Methods. (A) Blots were probed with anti-active Erk-1/-2 before stripping and reprobing with an antibody directed against total Erk-1/-2. (B) Blots were probed with anti-active JNK-1/-2 before stripping and reprobing with an antibody directed against total JNK-1. Representative of three isolates.

 
In contrast to our results with HUVEC [23], activation of Jun N-terminal kinase (JNK)-1/-2 was observed within 10 min after ICAM-1 cross-linking (Fig. 2B). This activity returned to basal levels by 1 h cross-linking. Treatment of VSMC with a combination of TNF and IL-1 for 15 min induced a large increase in activity of JNK-1/-2 (Fig. 2B).

Depletion of I{kappa}B{alpha} after ICAM-1 cross-linking was examined. As shown in Fig. 3 there was no depletion of I{kappa}B{alpha} after up to 1 h cross-linking. In contrast, treatment of VSMC with TNF/IL-1β for 15 min lead to a complete depletion of I{kappa}B{alpha} from the cytosol.


Figure 3
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  		Fig. 3 Depletion of I{kappa}B{alpha} after ICAM-1 cross-linking of VSMC. Resting VSMC from human pulmonary artery were treated with 15 µg/ml anti-ICAM-1 MAb for 30 min at 37°C, followed by RAM for the indicated times As negative controls they were treated with irrelevant antibody alone, anti-ICAM-1 MAb alone, RAM alone for 10 min. Cells were treated with 100 u/ml TNF and 100 u/ml IL-1β for 15 min as a positive control. Cells were lysed, resolved by SDS–PAGE and blotted onto nitrocellulose as described in Methods. Blots were probed with antibodies to I{kappa}B{alpha} before stripping and reprobing with anti-SM {alpha}-actin. Representative of three isolates.

 
3.3 Proliferation of VSMC after ICAM-1 cross-linking
Since VSMC have been shown previously to respond to a wide variety of stimuli with increased growth factor expression and increased proliferation, the effect of ICAM-1 cross-linking on these two responses was investigated. No increase in proliferation after ICAM-1 cross-linking for up to 72 h was observed, measured by [3H] TdR incorporation (Fig. 4). In contrast, incubation of serum starved VSMC with recombinant PDGF-BB for 24 h caused a significant increase in proliferation (P<0.001). No increase in mRNA for PDGF B chain, TGFβ1 or bFGF was observed after ICAM-1 cross-linking (data not shown).


Figure 4
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  		Fig. 4 Proliferation of VSMC after ICAM-1 cross-linking. Confluent monolayers of quiescent VSMC from human aorta were treated with 15 µg/ml anti-ICAM-1 MAb together with RAM for the indicated times or anti-ICAM-1 alone, an Ig depleted fraction of the anti-ICAM-1 preparation or 100 u/ml PDGF-BB for 24 h. 3H[TdR] was added to cells for the last 24 h of culture. Cells were fixed and lysed as described in Methods. The lysates were counted in scintillant for 1 min in a Canberra Packard Beta counter. Pooled data from three separate isolates.

 
3.4 Expression of VCAM-1 after ICAM-1 cross-linking in VSMC
To investigate the possibility that ICAM-1 ligation on the surface of VSMC leads to the induction of other cell adhesion molecules, RT-PCR was carried out on RNA extracts of ICAM-1 cross-linked cells, for full-length VCAM-1 mRNA, using specific intron-spanning oligodeoxynucleotide primers. Resting VSMC expressed low levels of VCAM-1 mRNA, however, after 6 h cross-linking an increase in VCAM-1 message was observed (Fig. 5A). Pre-treatment of VSMC with PD98059 abrogated its expression.


Figure 5
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  		Fig. 5 VCAM-1 expression after ICAM-1 cross-linking of VSMC. Quiescent VSMC from human pulmonary artery were treated with 15 µg/ml anti-ICAM-1 MAb for 30 min at 37°C, followed by RAM for the indicated times or anti-ICAM-1 MAb alone for 6 h, RAM alone for 6 h or 100 u/ml TNF for 6 h, ±pre-incubation with 30 µM PD98059 or 10 µM PSI. (A) RNA was extracted and subjected to RT-PCR with specific primers for VCAM-1 or β-actin. (B) Total cell protein (100 µg) was extracted and immunoprecipitated with anti-VCAM-1, followed by resolution by SDS–PAGE, Western blotting onto nitrocellulose and immunodetection with anti-VCAM-1, as described in Methods. Total protein (25 µg) was also resolved by SDS–PAGE, Western blotted and probed with anti-SM-{alpha}-actin, as a protein loading control. Representative of three isolates.

 
We examined whether the increase in VCAM-1 mRNA was accompanied by an increase in protein expression after ICAM-1 cross-linking in VSMC. One limitation of mimicking cell–cell interactions by cross-linking with monoclonal antibodies is the difficulty of examining cell surface proteins by flow cytometry or immunocytochemistry, due to unwanted reactions between the antibodies used to stain, with the RAM used to cross-link the cells. Thus, VCAM-1 was immunoprecipitated from whole cell extracts and immunodetection was conducted after resolution by SDS–PAGE, using MAbs directed against the same or different epitopes of VCAM-1. There was slight induction of VCAM-1 protein after treatment of VSMC with anti-ICAM-1 alone or RAM alone, but we observed a large increase in immunoprecipitated VCAM-1 after 12 h ICAM-1 cross-linking or after treatment of cells with TNF{alpha} for 6 h (Fig. 5B). Apart from 110-kDa, full-length VCAM-1 immunoprecipitated after ICAM-1 cross-linking, we also observed a lower molecular weight form of VCAM-1 of with an apparent molecular weight of 40–46 kDa, after ICAM-1 cross-linking. This truncated VCAM-1 molecule may correspond to the GPI-linked form of VCAM-1 described in the mouse [34–36] although further investigation is necessary (Fig. 5B). Pre-treatment of VSMC with the MEK inhibitor PD98059 caused abrogation of VCAM-1 induction (Fig. 5B), suggesting that ERK-1/-2 signalling is involved in ICAM-1 induced VCAM-1 expression. In contrast, PSI, an NF-{kappa}B inhibitor [29], had no effect, in agreement with our previous results [23].


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
VSMC within atherosclerotic lesions have been extensively studied and their phenotype is well defined. These cells acquire the ability to produce matrix and pro-inflammatory cytokines. They lose contractile function and are proliferative. There are, however, other cell types, which also contribute to the pathology of the disease, including the invading monocyte/macrophage and lymphocyte populations [1–3]. We have observed ICAM-1 expression on the surface of VSMC isolated by explants from aortic tissue and pulmonary artery tissue, and ICAM-1 positive VSMC have been observed within atherosclerotic lesions previously [10,13,15,16]. These cells also produce pro-inflammatory factors, and matrix-forming factors. It is likely that interactions between smooth muscle cells and the invading leukocytes have a role in maintaining the chronicity of the lesion. In order to mimic VSMC:leukocyte interactions, we ligated ICAM-1 on the surface of VSMC.

In agreement with other reports we observed an increase in phosphorylation of members of the mitogen activated protein kinase (MAPK) family after ICAM-1 cross-linking. VSMC were more sensitive than HUVEC since ICAM-1 MAbs did not need to be cross-linked in order to see activation of ERK-1/-2 cascades [23]). We also saw activation of JNK-1 after cross-linking, which has not been observed in HUVEC [23] but has been previously documented in rat brain MVEC [21], where ICAM-1 cross-linking induced JNK-1/-2 activity but not Erk-1/-2 activation. This suggests that there is cell type/species specific induction of MAPK pathways after ICAM-1 cross-linking. JNK-1/-2 activation has previously been associated with stress responses and induction of pro-inflammatory cytokine cascades [37,38]. Since ICAM-1 cross-linking on VSMC activates JNK-1, in vitro, we propose that this might provide an alternative pathway for the upregulation of pro-inflammatory mediators within atherosclerotic lesions and at sites of inflammation within the vasculature, where interactions between ICAM-1 expressing VSMC and leukocytes could occur. We are currently investigating whether ICAM-1 cross-linking leads to expression of pro-inflammatory cytokines.

A characteristic feature of the atherosclerotic lesion is that VSMC migrate from the media into the intima of the vessel. Here they re-enter the cell cycle and undergo substantial proliferation, thus contributing to the intimal thickening observed within the vessel wall [5,39]. VSMC used in this study were isolated by explant of cells from aorta or pulmonary artery, thus cells with a migratory capacity and consequently, possibly of the synthetic, proliferative phenotype were selected for. However, ICAM-1 ligation of explanted VSMC did not have any effect on their proliferation (Fig. 4). In contrast, previous work investigating the role of anti-MHC class I antibodies has shown that ligation of MHC class I with these antibodies can lead to increased proliferation of both vascular endothelial cells and VSMC [40–42]. This is accompanied by an increase in expression of growth factor receptors [43]. Interestingly, Hu et al. [44] have shown that Erk-1/-2 is involved in LDL-induced proliferation in VSMC isolated from atherosclerotic rabbit thoracic aortas. Interactions of ICAM-1 with ligands other than LFA-1, including fibrinogen [45], which is expressed within the lesion [46,47] have also been shown to activate Erks and to lead to cell proliferation [48,49], suggesting that pathways downstream of Erk activation may be different depending on the initial stimulus, in VSMC.

We looked for factors that may be involved in the continued leukocyte presence within the lesion. Increased expression of VCAM-1 was observed (Fig. 5). VCAM-1 expression on the surface of VSMC within atherosclerotic plaques has been well documented. Indeed, enhanced expression is observed on VSMC compared to endothelial cells [11,15]. It is possible that at least some of this expression may be due to interaction between infiltrating T cells and VSMC via ICAM-1 and LFA-1 interactions, as modelled in this paper. VCAM-1 would be expected to have pro-inflammatory effects via a number of different mechanisms, including retardation of very late antigen (VLA)-4 positive infiltrating monocytes/foam cells, possibly contributing to their activation through further integrin interactions. It is also possible that VCAM-1 can act as a co-stimulatory molecule as has been shown in some contexts [50], leading to activation of further T cell subsets expressed within the lesion.

In conclusion, this paper describes a possible mechanism whereby ligation of ICAM-1 on VSMC leads to upregulation of VCAM-1, which might, in vivo, contribute to the cycle of pro-inflammatory events within the atherogenic vessel wall.

Time for primary review 15 days.


   Acknowledgements
 
This work was supported by the British Heart Foundation. The authors are grateful to Ms. Ginette Hoare for helpful discussions and technical advice, and to Ms. Angela Holder for technical assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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