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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:784-793

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:784-793.)
© 1999 American Heart Association, Inc.

Original Contributions

Enzymatically Modified, Nonoxidized LDL Induces Selective Adhesion and Transmigration of Monocytes and T-Lymphocytes Through Human Endothelial Cell Monolayers

Mariam Klouche; Andreas E. May; Monika Hemmes; Martina Meßner; Sandip M. Kanse; Klaus T. Preissner; Sucharit Bhakdi

From the Institute of Medical Microbiology (M.K., M.H., M.M., S.B.), Johannes Gutenberg University of Mainz; Haemostasis Research Unit (A.E.M., S.M.K., K.T.P.), Max Planck Institute, Bad Nauheim; and I. Med. Klinik und Deutsches Herzzentrum (A.E.M.), Technische Universität München, Germany.

Correspondence to Dr Mariam Klouche, Institute of Medical Microbiology, Johannes-Gutenberg University of Mainz, Obere Zahlbacher Straße, 55101 Mainz, Germany. E-mail Klouche{at}mail.Uni-Mainz.de


*    Abstract
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Abstract—Circulating monocytes and T lymphocytes extravasate through the endothelium at sites of developing atheromatous lesions, where they tend to accumulate and mediate the progression of the disease. We have previously demonstrated the presence of an enzymatically degraded, nonoxidized form of LDL (E-LDL) in early human fatty streaks, which possesses major biological properties of an atherogenic lipoprotein. The effects of E-LDL on human endothelial cells have now been studied with respect to adhesion and transmigration of monocytes and T lymphocytes. E-LDL induced a rapid and dose-dependent selective adhesion of monocytes and T lymphocytes to endothelial cell monolayers within 30 minutes of incubation. Maximal increases in the number of adherent monocytes (8-fold) and of adherent T lymphocytes (4-fold) were observed after treatment with 50 µg/mL E-LDL. E-LDL was more active than oxidized LDL (ox-LDL), whereas native LDL produced only minor adhesive effects. Both E-LDL and ox-LDL enhanced transmigration of monocytes and of T lymphocytes through endothelial monolayers. Again, E-LDL was more potent than ox-LDL, inducing transmigration to a similar extent as N-formyl-Met-Leu-Phe. In endothelial cells, E-LDL stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cells adhesion molecule-1 (PECAM-1), P-selectin, and E-selectin with distinct kinetics. Analyses with blocking antibodies indicated that ICAM-1 and P-selectin together mediated approximately 70% of cell adhesion, whereas blocking of PECAM-1 had no effect on adhesion but reduced transmigration to less than 50% of controls. E-LDL also upregulated expression of ICAM-1 in human aortic smooth muscle cells, and this correlated with increased adhesion of T lymphocytes. E-LDL is thus able to promote the selective adhesion of monocytes and T lymphocytes to the endothelium, stimulate transmigration of these cells, and foster their retention in the vessel wall by increasing their adherence to smooth muscle cells. These findings underline the potential significance of E-LDL in the pathogenesis of atherosclerosis.


Key Words: atherogenesis • adhesion • transmigration • LDL • T lymphocytes • monocytes • endothelium


*    Introduction
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Increased adherence of leukocytes to the endothelium and their transmigration into the arterial wall is one of the earliest events in atherogenesis. Immunohistochemical investigations have demonstrated the presence of monocytes/macrophages in human early atherosclerotic lesions,1 and the kinetics of adherence and emigration of blood-borne monocytes into developing lesions have been lucidly analyzed in experimental animals.2 3 4 5 6 In addition to monocytes, T lymphocytes also accumulate in the human atherosclerotic intima.7 8 9 10 It has been estimated that approximately 80% of cells in the early lesion are monocytes/macrophages, and 10% to 20% are T lymphocytes.11 Directed leukocyte trafficking is regulated by a coordinate expression of adhesion molecules and by locally generated chemokines, the latter acting by direct chemoattractant effects and by regulating the affinity of integrins for their ligands.12 13 14 15 Accordingly, monocyte and T-lymphocyte recruitment into early atheromatous lesions must involve changes in endothelial adhesiveness for these cells. Enhanced attachment of monocytes to endothelial cells (ECs) has been observed in vitro after exposure of the cells to oxidized LDL (ox-LDL)16 17 18 and to VLDL,19 20 and ox-LDL has been shown to mediate upregulation of adhesion molecules in these cells.18 21 22 23 Schwartz et al24 demonstrated that the enhanced binding of monocytes to ECs treated with minimally modified LDL was mediated by the selective induction of chemokines. Furthermore, ox-LDL induced production of chemokines in monocytes/macrophages, and enhanced transmigration of monocytes through endothelial–smooth muscle cell cocultures after treatment with ox-LDL,25 26 a process that was mainly attributable to the induction of monocyte chemoattractant protein-1 (MCP-1).27

Although ox-LDL is generally considered to be a major agent responsible for initiating and sustaining the pathological process in atherosclerosis, we are considering another type of LDL modification to be of importance. We have found that nonoxidative, enzymatic degradation of LDL (E-LDL) by lysosomal enzymes transforms this, but not other lipoproteins, to a moiety that rivals ox-LDL in its atherogenic potential.28 E-LDL is rapidly internalized by macrophages by means of a scavenger receptor-dependent pathway. Macrophage foam cells thereby produce and release large quantities of MCP-1 in the virtual absence of interleukin-1 (IL-1), IL-8, and tumor necrosis factor-{alpha} (TNF-{alpha}) production.29 At high concentrations, E-LDL is cytotoxic to human macrophages. E-LDL displays the same micromorphology as lipid droplets that have been isolated from atherosclerotic lesions,30 and E-LDL activates complement in vitro.28 Complement activation is in fact a prominent, consistent feature of the atherosclerotic lesion.30 31 32 An important role for complement in lesion development is indicated by the fact that complement C6-deficient rabbits are markedly protected against development of diet-induced atherosclerosis.33 Direct evidence for the presence of E-LDL in early human lesions has been obtained by the use of specific monoclonal antibodies that recognize epitopes on E-LDL, but not on native or ox-LDL.34 Extensive extracellular accumulation of E-LDL was observed in colocalization with activated complement at the earliest stages of lesion development. In contrast, ox-LDL is only sparsely present in early lesions, does not activate complement, and differs micromorphologically from E-LDL and from LDL derivatives isolated from atheromas. For these reasons, we are considering that LDL may be transformed to an atherogenic moiety predominantly by combined enzymatic alteration rather than solely by oxidation.

We questioned whether E-LDL might promote adhesion of blood cells and induce their transmigration through endothelial cell monolayers. The affirmative results further support our alternative hypothesis on the pathogenesis of atherosclerosis.


*    Methods
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Isolation and Culture of Endothelial and Smooth Muscle Cells
Primary human ECs were isolated from umbilical cord veins using collagenase/dispase and were cultivated in EC growth medium (PromoCell) at 37°C in 5% CO2. The medium was supplemented with 1% FCS, 0.5 ng/mL human recombinant basic fibroblast growth factor-ß, 0.5 ng/mL human recombinant epidermal growth factor, 25 mg/L gentamycin, and 1.25 mg/L amphotericin B. In addition to the typical cobblestone morphology, purity of ECs was ascertained by positive staining for von Willebrand factor (rabbit anti-human vWF, Sigma). Smooth muscle cells (SMCs) were obtained from pieces of human aortas obtained during aneurysm surgery (7 donors, mean age, 72 years; range, 45 to 87 years; 6 men, 1 woman) by courtesy of Dr W. Schmiedt, Department of Heart and Thoracic Surgery, University of Mainz. Media fragments from the human aorta were prepared, and SMCs were obtained by outgrowth35 in medium containing 1 ng/mL human recombinant basic fibroblast growth factor-ß, 5 ng/mL human recombinant epidermal growth factor, 25 mg/L gentamycin, and 1.25 mg/L amphotericin B. The purity of SMCs was evaluated by staining with a monoclonal antibody directed against SMC-specific {alpha}-actin (clone 1A4, Sigma). The explants were incubated at 37°C in 5% CO2 in a humidified atmosphere. Medium was changed every 3 days for both ECs and SMCs. Twenty-four hours before the experiments, ECs and SMCs were kept in DMEM (Gibco BRL) without any additives. All experiments with ECs or SMCs were performed before passage 5.36

Isolation of Leukocytes
Human monocytes depleted of platelet contaminants were isolated from buffy coats of healthy blood donors (courtesy of the University of Mainz Blood Bank) by density-gradient separation.37 Purity was >95% as determined by flow cytometry, and viability was >95% as determined by trypan blue exclusion. In addition, human myelomonocytic HL-60 cells (German Collection of Microorganisms and Cell Cultures), which were maintained in RPMI medium supplemented with 10% FCS, 1 mmol/L glutamine, 1% sodium pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco-BRL), were used. Medium was changed every 3 days. Twenty-four hours before the experiments, monocyte differentiation was induced by addition of 1-{alpha}-25-dihydroxy-vitamin D3 (50 ng/mL) and transforming growth factor-ß (TGF-ß) (1 ng/mL), resulting in a monocyte-specific antigen expression pattern for CD14, CD11b, CD18, and CD87, as determined by flow cytometry (data not shown).

T lymphocytes were isolated using Ficoll density-gradient centrifugation followed by formation of rosettes with sheep red blood cells (Virion). Contaminating erythrocytes were removed by treatment with lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L KHCO3, 0,1 mmol/L EDTA). Viability of T lymphocytes was >99% by trypan blue exclusion and purity was >97%.

For adhesion or transmigration experiments, isolated leukocytes were adjusted to 1x106 cells/mL in DMEM.

LDL Isolation
LDL was isolated from human fasting plasma by preparative ultracentrifugation using KBr gradients according to a modified protocol by Lindgren et al.38 Plasma was obtained from healthy blood donors lacking hypertension, diabetes, or signs of ischemic heart disease and not taking any medication. To prevent artificial oxidation of LDL, it was dialyzed against Tris-NaCl buffer containing 1 mmol/L EDTA and stored at 4°C for up to 2 weeks. The cholesterol content was determined using an enzymatic test (Boehringer-Mannheim), and the protein content was analyzed using the Bradford method (Roth).

Lipoprotein Modifications
Enzymatic and oxidative modifications of native LDL were conducted as described.28 Briefly, LDL was treated with trypsin (6.6 µg/mL, Sigma), cholesterol esterase (40 µg/mL, Boehringer-Mannheim), and neuraminidase (79 mU/mL, Behring). For extensive oxidative modification of LDL, CuSO4 (50 µmol/L) was used according to the classic protocols.39 The absence of oxidation products in E-LDL was verified by the determination of thiobarbituric-reactive substances (TBARS) to quantify lipid peroxidation products. Modified lipoproteins were stored at 4°C and used within a week. During LDL preparation and subsequent modification, general precautions were taken to avoid LPS contamination, and regular control assays with the Limulus endotoxin assay (E-Toxate, Sigma) yielded negative results.

Adhesion Assay
For adhesion studies, ECs or SMCs were seeded in 96-well dishes and allowed to proliferate to confluence (approximately 48 hours). After 24-hour growth in DMEM without additives, EC or SMC monolayers were stimulated with native LDL, E-LDL, or ox-LDL at 37°C. Cells incubated with medium alone, with LPS (1 µg/mL), or with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL) served as negative, positive, or positive controls, respectively. In addition, medium containing the enzyme-mix alone was used, which did not result in any alteration of monocyte or T-lymphocyte adhesion. After rinsing twice with warm, serum-free medium, 5x105 monocytes, HL-60 cells, or T lymphocytes were added to each well. After the stated time of incubation, nonadherent leukocytes were rinsed off twice with DMEM. This procedure did not affect the integrity of the monolayers as determined by phase-contrast microscopy. The number of attached leukocytes was determined using three different methods. (1) The number of adherent leukocytes was counted in each of 10 high-power fields. (2) For screening a large number of conditions, the leukocytes were labeled with the nontoxic chromophore 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein, acetomethyl ester (BCECF-AM, Molecular Probes) at 5 µmol/L for 30 minutes at 37°C before incubation with lipoprotein-treated EC or SMC monolayers. BCECF-AM has no deleterious effects on leukocyte function and additionally serves as an indicator for cell viability. Fluorescent-labeled leukocytes were washed twice in PBS to remove nonincorporated fluorochrome and resuspended in DMEM to a final concentration of 1x106/mL. After termination of the adhesion assay, nonadherent cells were rinsed off, and the remaining, firmly adherent leukocytes were lysed with 0.5N NaOH. Fluorescence was determined in a fluorescence plate reader (Titertec, Fluoroscan II; excitation 485 nm, emission 538 nm). (3) In some experiments, monocytes and monocyte-like cells were labeled with [3H]thymidine, and the number of adherent cells was determined by counting the solubilized leukocytes in a ß-counter (Beckman Instruments, LS6000 TA). Results obtained with this assay were essentially the same as with the fluorimetric method or optical counting. For fluorochrome or radioactive adhesion assays, standard curves with known numbers of labeled leukocytes were tested in parallel.

Transmigration Assays
For transmigration studies, we used a similar system as that described by Taylor et al.40 Human ECs (2x105/cm2) were seeded onto polycarbonate membranes (5.0-µm pore size) of transwell inserts closely fitted into 24-well plates (Nunc) and allowed to grow for a further 48 hours. Then, medium containing E-LDL, ox-LDL, or LDL at 50 µg/mL was introduced beneath the filter supporting the EC monolayer for 4 hours. The lipoproteins were thus present in the subendothelial space, which would most closely resemble in vivo conditions. Medium alone, or medium containing fMLP (1x10-8 mol/L) or LPS (1 µg/mL), served as negative or positive controls, respectively. Incubation of endothelial cells with the enzyme mix alone, which was used to generate E-LDL, had no effect on the transmigration of monocytes or T lymphocytes. For T-lymphocyte analyses, the medium containing E-LDL had to be removed after the 4-hour endothelial stimulation because subsequent exposure of T lymphocytes to E-LDL resulted in cytotoxic effects that created artifacts. For migration studies, 1x106/mL fluorochrome-labeled leukocytes (5x105/well) were presented to the endothelial side of the filter in the absence or presence of stimuli in lower or subendothelial compartment (trans) and allowed to migrate through the filter for 90 minutes. Thereafter, the medium below the filter was recovered, and the leukocytes that had migrated through the EC monolayer and those that were removed from the lower filter surface by gentle scraping with a rubber policeman were quantified by counting or by determination of total fluorescence. Because the integrity of the endothelial monolayer is crucial for the reliability of the assay, filters were stained with Giemsa after completion of the assays, and only those wells that showed intact endothelial cell monolayers were evaluated. In addition, selected filters were embedded in epoxy-resin, cut in a microtome, and evaluated microscopically.

Expression of Adhesion Molecules
ECs were plated in 96-well microtiter wells and subjected to treatment with E-LDL, ox-LDL, LDL, or LPS (1 µg/mL) for 30 minutes to 4 hours. Supernatants were then discarded, and the cells were washed 3 times with cold PBS. Incubation with fluorescein-labeled mouse anti-human ICAM-1 (CD54, R&D), platelet-endothelial cells adhesion molecule-1 (PECAM-1) (CD31), E-selectin (CD62E), or P-selectin (CD62P) (all from Camon) was conducted for 1 hour. As a negative control, an isotype-matched antibody against an irrelevant antigen was used. After washing twice with ice-cold PBS, EC were lysed with 0.5N NaOH and the fluorescence intensity was determined in a Fluoroscan as described for the adhesion assays.

Blocking Experiments
To determine the contribution of individual adhesion molecules to the interaction of leukocytes with lipoprotein-treated EC or SMC, blocking antibodies against ICAM-1, PECAM-1, E-selectin and P-selectin as well as blocking anti-CD11a (all from Camon) and anti-CD18 (Ancell) antibodies were used. Initially, the optimal blocking concentration was selected from dose-response curves performed for each antibody, which was 1:200 for ICAM-1, PECAM-1, and E-selectin and 1:100 for P-selectin. After stimulation of EC or SMC monolayers with the different lipoprotein preparations for 4 hours and 2 washes with DMEM, blocking antibodies directed against the adhesion molecules were applied to the cells for 1 hour at 37°C. Antibodies against the leukocyte function antigens CD11a and CD18 were incubated at 50 µg/mL with monocytes or T lymphocytes. After washing in DMEM, the labeled leukocytes were added to ECs, and adhesion was quantified after 90 minutes, as described.

Statistical Analysis
The Kruskall-Wallis test was used to determine the significance of differences in groups with more than 2 variables. To compare differences between control values and the different stimulants, the Mann-Whitney U test was used. Differences were considered significant at P<0.05. The results were expressed as mean±SD. Analysis was performed with SPSS software (SPSS Inc).


*    Results
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 *Results
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Monocyte Adhesion to Cultured Human Endothelial Cells
Treatment of human primary ECs with E-LDL (1 to 100 µg/mL) induced a dose-dependent increase in the number of firmly adherent human blood monocytes (Figure 1Down, left) and of the human monocyte-like HL-60 cells (Figure 1Down, right). Monocytes or HL-60 cells were added at an excess of cells (5x105 per well), which is equivalent to 7.7±0.8 cells per EC, based on an average number of 6.5±0.7x104 EC per well (mean±SD, n=6). Under unstimulated control conditions, monocyte adhesion was comparably low, reaching 2% to 9% of monocytes or HL-60 cells added. Maximal adhesion was observed after pretreatment of ECs with 50 µg/mL E-LDL for 2 hours, which produced an 8-fold increase in the number of adherent monocytes compared with untreated control cells. A significant elevation of monocyte adhesion to ECs was observed in the presence of only 10 µg/mL E-LDL. E-LDL was significantly more active than ox-LDL in stimulating adhesion of human blood monocytes to pretreated ECs (increase by a factor of 8.0 versus 4.8; P<0.01). In contrast, both E-LDL and ox-LDL were equally effective in promoting HL-60 cell adhesion. Incubation of ECs with native LDL did not induce monocyte or HL-60 cell adhesion to the monolayers.



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  		Figure 1. Dose-dependent adhesion of monocytes and HL-60 cells to ECs treated with E-LDL, ox-LDL, or native LDL. Confluent monolayers of ECs were pretreated with various concentrations of lipoproteins for 2 hours, and firm adhesion of monocytes (left panels) or HL-60 cells (right panels) was quantified after incubation for 1 hour. The results represent mean values of 6 individual experiments for monocytes and 4 separate experiments for HL-60 cells±SD, with each experimental condition performed in triplicate; *differences were significant at P<0.05.

Monocyte adhesion was substantially stimulated after only 30 minutes of exposure of ECs to 50 µg/mL E-LDL (Figure 2Down). Adhesion was maximal at 2 hours of E-LDL treatment and was sustained at near maximal levels for at least 8 hours. Kinetic studies confirmed that E-LDL was superior to ox-LDL in stimulating the adhesion of human peripheral blood monocytes (Figure 2ADown), whereas similar adhesion kinetics of HL-60 cells were observed for E-LDL and ox-LDL (Figure 2BDown). Monocyte adhesion remained unaltered when ECs were exposed to native LDL. Treatment of ECs with cycloheximide (1 µg/mL) 2 hours before the addition of E-LDL markedly reduced monocyte binding, suggesting that protein synthesis was necessary for optimal induction of monocyte adhesion (data not shown). Dose-response curves showed that at the concentration tested, cycloheximide was not toxic for ECs and also did not result in an observable cell loss or disruption of the monolayer.



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  		Figure 2. Time course of monocyte and HL-60 cell adhesion to lipoprotein-treated ECs. After pretreatment of ECs for 1 hour with E-LDL ({bullet}), ox-LDL ({blacksquare}), or native LDL ({blacktriangleup}), monocytes (A) and HL-60 cells (B) were allowed to adhere for various periods of time. The results represent mean values of 8 individual experiments for monocytes and 5 individual experiments for HL-60 cells±SD, with each experimental condition performed in triplicate. Each experiment was performed with different ECs.

Adhesion of T-Lymphocytes to E-LDL-Treated Endothelial Cells
Similar to monocytes, adhesion of T-cells was dose-dependently increased by pretreatment of ECs with E-LDL (Figure 3ADown). T-cells were added at an excess of cells (5x105 per well) yielding a similar ratio as determined for monocyte adhesion assays of approximately 7.7 T-cells per EC. Adhesion under control conditions was in the range of 2% to 8% of T-cells added. However, the extent of T-lymphocyte adhesion was lower compared with monocytes. In the presence of 5 µg/mL E-LDL, the number of T lymphocytes adhering to the endothelial monolayer nearly doubled. Maximal effects were observed at 25 µg/mL E-LDL at which concentration the number of T lymphocytes increased 4.5-fold. Enhanced binding of T lymphocytes began after only 15 minutes of EC exposure to E-LDL and reached maximal levels after 30 minutes of incubation (Figure 3BDown). Binding was sustained for at least 8 hours. As opposed to the selective effect induced by modified lipoproteins, LPS stimulated a nonselective adhesion of monocytes and T-cells to pretreated ECs (data not shown).



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  		Figure 3. Pronounced adhesion of T lymphocytes to E-LDL-treated ECs. A, After pretreatment of ECs with different doses of E-LDL for 1 hour, adherent T lymphocytes were quantified. B, After pretreatment of ECs with E-LDL (10 µg/mL), T-lymphocyte adhesion was measured for various periods of time. The results represent mean values of 4 individual experiments±SD, with each experimental condition performed in triplicate. Different ECs were used for each experiment.

E-LDL Stimulates Transmigration of Monocytes and T-Lymphocytes Through Endothelial Monolayers
EC monolayers were grown on polycarbonate membranes, and stimuli were introduced into the subendothelial compartment for 4 hours. Then, monocytes or T lymphocytes were added to the upper compartment and transmigration was allowed to proceed for 1.5 hours. E-LDL induced a dose-dependent stimulation of both monocyte and T-lymphocyte transmigration (Figure 4Down). Maximal transmigration of monocytes with a 6.2-fold increase was observed with 50 µg/mL E-LDL (Figure 4ADown). Migration of T lymphocytes was less pronounced but still was significantly augmented approximately 3-fold compared with the migration observed in untreated control wells (Figure 4BDown).



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  		Figure 4. Transmigration of monocytes and T lymphocytes through E-LDL-treated ECs. EC monolayers were grown on transwell filters and stimulated with various concentrations of E-LDL that were presented in the subluminal compartment for 4 hours. Untreated ECs that were kept in medium alone served as controls for spontaneous migration. After labeled leukocytes (5x105/well) were allowed to migrate from the luminal side through the EC monolayer for 90 minutes, filter-associated and subluminal compartment monocytes (A) or T lymphocytes (B) were quantified. Results are expressed as percent of transmigrating cells relative to total leukocytes as determined in 3 individual experiments±SD, with each experimental condition performed in triplicate; *differences were significant at P<0.05.

The effect of E-LDL on monocyte and T-lymphocyte transmigration was comparable with that induced by the classic chemoattractant fMLP and by LPS (Figure 5Down). Particularly with regard to stimulation of monocyte transmigration, E-LDL appeared to be more effective than ox-LDL applied at the same concentration (50 µg/mL) (increased by a factor of 6.2 versus 4.1; P<0.01) and was also more effective in enhancing T-lymphocyte transmigration (increased by a factor of 3.3 versus 2.0; P<0.05). Incubation of endothelial monolayers with native LDL did not significantly enhance monocyte or T-lymphocyte migration.



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  		Figure 5. Comparison of E-LDL with other agonists in their potency to stimulate monocyte transmigration. After stimulation of EC monolayers with E-LDL, ox-LDL, or native LDL (each at 50 µg/mL cholesterol), LPS (1 µg/mL), or fMLP (1x10-8 mol/L) for 4 hours, transmigration of monocytes (A) or T lymphocytes (B) was quantified as described for Figure 4Up. Results are expressed as percent of transmigrated cells in 4 different experiments±SD, each with triplicate cultures for each experimental condition; *differences were significant at P<0.05.

Leukocyte Adhesion to Human Smooth Muscle Cells
To exert their pathological role in atherogenesis, after transmigration infiltrating leukocytes must be retained in the intima. In addition to their interaction with extracellular matrix components, leukocytes may adhere to intimal SMCs. We evaluated the adhesion of monocytes and T lymphocytes to human aortic SMCs that had been pretreated with different concentrations of E-LDL, ox-LDL, or native LDL (Figure 6Down). As opposed to EC, treatment of SMCs with both E-LDL and ox-LDL induced a more pronounced adhesion of T lymphocytes compared with monocytes. Again, the effect of E-LDL treatment was significantly stronger than that induced by ox-LDL. After a 4-hour incubation with only 25 µg/mL E-LDL, adhesion of T lymphocytes to SMCs was enhanced 6-fold. Treatment of SMCs with native LDL had no effect on leukocyte adhesion.



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  		Figure 6. Adhesion of monocytes and T lymphocytes to E-LDL-stimulated human aortic SMCs. After incubation of SMCs with various concentrations of E-LDL (A), ox-LDL (B), or native LDL (C) for 4 hours, adherence of T lymphocytes ({blacksquare}) or monocytes ({bullet}) was quantified. Results are expressed as adhesion ratio calculated in comparison with spontaneous adhesion of leukocytes to untreated control SMCs. Each point represents mean values of 7 different experiments±SD, each with triplicate cultures for each experimental condition. Each experiment was performed with 2 different SMC preparations.

Distinct Pattern of Adhesion Molecule Expression by Endothelial and Smooth Muscle Cells
The induction of adhesion molecule expression was evaluated with a cell ELISA (Figure 7Down), and the results obtained with ECs (Figure 7Down, left) and SMCs (Figure 7Down, right) after stimulation with E-LDL for 4 hours are shown. In ECs, constitutive expression of ICAM-1 was stimulated in the presence of 10 µg/mL E-LDL and maximal ICAM-1 expression was observed at 50 µg/mL, resulting in a 3.8-fold increase. Similarly, E-selectin expression peaked at 50 µg/mL (fluorescence intensity 340±44) compared with unstimulated ECs. Most pronounced was the induction of PECAM-1 expression on ECs, which reached 5-fold values over baseline and was observed over a broad range of concentrations. P-selectin expression was less pronounced, to approximately 2-fold values.



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  		Figure 7. Expression of adhesion molecules after E-LDL treatment of ECs or SMCs. After pretreatment of ECs (left) or SMCs (right) with various concentrations of E-LDL for 4 hours, the expression of ICAM-1, PECAM-1, E-selectin, and P-selectin was quantified by cell ELISA. Results are mean values of 4 distinct experiments±SD, each with triplicate cultures for each experimental condition; *differences were significant at P<0.05.

Substantial induction of ICAM-1 expression was observed on SMCs. Here, only 10 µg/mL E-LDL produced maximal ICAM-1 expression (factor of 4 over baseline). As expected, the endothelial markers PECAM-1 and P-selectin were not expressed by SMCs. There was a slight but consistent stimulation of E-selectin in the presence of low E-LDL concentrations.

Kinetics of Adhesion Molecule Expression by Endothelial and Smooth Muscle Cells
Expression of adhesion molecules provoked by E-LDL followed distinct kinetics. Endothelial expression of P-selectin was elevated as early as 15 minutes after E-LDL application and slowly declined after 30 minutes (Figure 8ADown). Enhancement at 15 minutes was 4- to 5-fold, and still approximately 2-fold at 4 to 8 hours. Unlike P-selectin, other adhesion molecules appeared later in the course of E-LDL stimulation. ICAM-1 and PECAM-1 expression increased after 4 hours of E-LDL incubation, was maximal within 8 hours, and then gradually declined. At 24 hours, levels remained slightly above baseline. In contrast, E-selectin showed an increase at 4 hours and returned to baseline at 24 hours.



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  		Figure 8. Kinetics of adhesion molecule expression in ECs and SMCs after treatment with E-LDL. ECs (A) or SMCs (B) were exposed to E-LDL for various periods of time, and the expression of ICAM-1 ({blacksquare}), PECAM-1 ({bullet}), P-selectin ({square}), and E-selectin ({circ}) was measured by cell ELISA. Results are expressed as mean fluorescence and represent mean values of 3 distinct experiments±SD, each with triplicate cultures for each experimental condition.

In SMCs, ICAM-1 expression peaked at 4 hours and still showed elevated levels at 8 hours of E-LDL incubation (Figure 8BUp). Expression of E-selectin was only slightly augmented at 4 hours. As controls, the EC markers PECAM-1 and P-selectin were determined, and they remained unaltered during the 24-hour incubation period.

Comparison of Endothelial Cell and Smooth Muscle Cell Adhesion Molecule Expression by E-LDL, Ox-LDL, or Native LDL
To evaluate adhesion molecule expression induced by differently modified LDL, EC or SMC monolayers were stimulated with E-LDL, ox-LDL, or native LDL for 4 hours (Figure 9Down). Because of the different susceptibility to E-LDL, ECs (Figure 9ADown) were stimulated with 50 µg/mL, whereas SMCs were incubated with 10 µg/mL (Figure 9BDown). Both E-LDL and ox-LDL were equally effective in increasing endothelial expression of ICAM-1, E-selectin, and P-selectin. E-LDL was significantly more effective in the induction of PECAM-1 expression compared with ox-LDL. Incubation of ECs with native LDL resulted in a minor elevation of ICAM-1 expression, and the other adhesion molecules remained unaltered. In SMCs, E-LDL increased expression of ICAM-1 to a greater extent than ox-LDL. A slight induction of E-selectin expression was observed with both E-LDL and ox-LDL. Native LDL did not affect adhesion molecule expression in SMCs.



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  		Figure 9. Induction of adhesion molecule expression by E-LDL, ox-LDL, or native LDL. After pretreatment of ECs (A) or SMCs (B) with E-LDL (black bars), ox-LDL (gray bars), or native LDL (white bars), the expression of adhesion molecules as indicated was measured by cell ELISA. Treatment was conducted for 4 hours at 50 µg/mL for ECs and at 10 µg/mL for SMCs. Results represent mean values of 3 different experiments±SD, each with triplicate cultures for each experimental condition; *differences were significant at P<0.05.

Functional Analysis of Adhesion Molecules on Endothelial Cells
The contribution of ICAM-1, PECAM-1, P-selectin, and E-selectin to monocyte adhesion (Figure 10Down, black bars) and transmigration (Figure 10Down, white bars) was investigated by incubation of E-LDL-stimulated ECs (50 µg/mL) with blocking antibodies. ICAM-1 was the single most important molecule responsible for monocyte adhesion because blocking of ICAM-1 reduced monocyte adhesion to ECs by approximately 60%. Additional blocking of P-selectin produced only a minor further reduction of adhesion that was not influenced in the presence of any third antibody. Blocking of E-selectin or PECAM-1 did not significantly reduce monocyte adhesion. Notably, blocking antibodies to PECAM-1 reduced transmigration to less than 50% of the control.



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  		Figure 10. Contribution of adhesion molecules for E-LDL-induced monocyte adhesion and transmigration. After stimulation of ECs with E-LDL (50 µg/mL) for 4 hours and incubation with blocking antibodies directed against adhesion molecules as indicated for 1 hour, monocyte adhesion (black bars) or transmigration (white bars) was performed and quantified as described. Results are means of 3 different experiments±SD, each with triplicate cultures for each experimental condition; *differences were significant at P<0.05.


*    Discussion
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 *Discussion
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This study demonstrates that E-LDL induces the expression of a distinct pattern of adhesion molecules and selectively increases the adhesive properties of human primary ECs and of human aortic SMCs for monocytes and T lymphocytes. Dose- and time-dependent increases in the number of adherent blood monocytes and T lymphocytes to E-LDL-treated ECs were noted. These effects were also found with monocyte-like human HL-60 cells. Increases in the adhesive properties of the endothelium are thought to be crucial for the initiation of atherosclerosis.41 In experimental animals, prelesional nascent stages are indeed characterized by preferential adherence and intimal penetration of blood-borne monocytes.4 5 In addition to monocytes, T lymphocytes are present in human early atherosclerotic lesions.7 9 10 11 42 43 Our in vitro data show that adhesion of both cell types significantly increased as early as 30 minutes after exposure of ECs to E-LDL. In the absence of cytotoxic effects on ECs, cycloheximide blocked the increase in leukocyte binding, suggesting that E-LDL may induce the synthesis of specific adhesion proteins. However, the relatively rapid increase of leukocyte adhesion observed indicates that mechanisms other than de novo protein synthesis may be involved. In accordance with published data, ox-LDL also enhanced monocyte and T-lymphocyte adhesion to ECs, the effects being somewhat less pronounced than those induced by E-LDL. The magnitude of monocyte–endothelial interactions induced by ox-LDL was comparable with that observed by other investigators.16 24 44 Similar to our results with E-LDL, minimally modified LDL induced monocyte but not neutrophil adhesion to ECs (data not shown), whereas LPS induced adhesion of both cell types.44 LDL modified by incubation with EC-SMC cocultures also did not induce increased adhesion of neutrophils.27 The reduced adhesion of monocytes and T lymphocytes to ECs or SMCs treated with high concentrations of E-LDL (>100 µg/mL cholesterol) is likely to be explained by a cytotoxic effect of E-LDL (data not shown). At concentrations up to 50 µg/mL E-LDL, however, no significant cytotoxic effects occurred even after prolonged exposure for up to 24 hours.

In the natural course of lesion progression, adhesion of leukocytes to the endothelium is followed by transmigration into the subendothelial space. Our data show that E-LDL not only increased the selective adhesion of monocytes and T lymphocytes, but also promoted their transmigration through EC monolayers. The enhancement of monocyte transmigration was more pronounced than that of T lymphocytes and was comparable to the migration induced by fMLP. Accentuated transmigration of monocytes has previously been observed in a coculture model of ECs and SMCs treated with cell-modified LDL.26 27 Enhanced migration of monocytes was shown to occur in an animal model of diet-induced atherosclerosis as well.2 3 These authors demonstrated that more than 90% of monocyte migration induced by cell-modified LDL was attributable to the induction of MCP-1 by EC-SMC cocultures.27 That MCP-1 induction by minimally modified LDL was required for stimulation of monocyte-EC adhesion was also observed by Berliner et al.44 We have shown previously that E-LDL potently and selectively stimulates the expression MCP-1 by human macrophage foam cells29 ; thus MCP-1 may also be a causative factor in our model.

Migration of T-cells in developing atheromatous lesions has not been systematically addressed in vitro. We analyzed human peripheral T-cells in the same transmigration model as established for blood monocytes. A significant enhancement of T-cell adhesion to E-LDL-pretreated ECs and enhanced transmigration were apparent. Our results are reminiscent of vital observations showing large numbers of T-cells in human prelesional areas, fatty streaks, and cap regions of full-grown atheromas.10 11 42 43 Similar findings have been made with cholesterol-fed experimental animals.45

The selective recruitment of monocytes and T-cells to the subendothelial space is likely to be the result of a local induction of adhesion molecules and chemotactic factors by lesional atherogenic LDL. Indeed, enhanced local expression of adhesive molecules including ICAM-1 and E-selectin has been detected in human atheromatous lesions.46 47 The present study was restricted to an analysis of adhesion molecules, and the potential significance of chemokine production has yet to be defined. We observed a significant induction of ICAM-1, PECAM-1, E-selectin, and P-selectin, of which particularly ICAM-1 and P-selectin were required for the maximal mediation of monocyte adhesion. These results compare well with recent data by Duplaa et al47 showing a marked increase in the expression of ICAM-1 and E-selectin on ECs adjacent to previously recruited macrophages in human atheromatous lesions. In fact, enhanced circulating EC adhesion molecules were observed in patients with atherosclerosis, and soluble ICAM-1 has been suggested as a marker for EC activation in atherosclerosis.48 In addition to ICAM-1/ß2-integrin–mediated adhesion of monocytes, vascular cell adhesion molecule-1 (VCAM-1)/VLA4 interactions contribute to firm adhesion of leukocytes. We did not address this additional mechanism of regulation, which is known to be involved in ox-LDL-mediated adhesion of monocytes to ECs. In contrast, experiments with blocking antibodies indicated that PECAM-1 might be more important for monocyte transmigration, as had been shown for granulocyte trafficking.49 However, interactions of high-affinity Fc receptors on monocytes with the Fc portions of blocking antibodies may also be responsible for the lack of effect on adhesion by anti-PECAM-1 antibodies and the unexpectedly minor blocking effects produced by antibodies directed against E-selectin and P-selectin. Therefore, the possibility that the adhesion effects observed are mediated by Fc receptors is not excluded by the methods used. In line with our results, E-selectin was observed on ECs in atherosclerotic lesions with immunohistochemistry in vivo.50 The E-LDL-mediated upregulation of P-selectin may reflect its rapid transfer from internal stores to the cell surface. In contrast, the later and more sustained expression of ICAM-1 and PECAM-1 is likely because of a de novo synthesis induced by E-LDL, which is supported by the lack of adhesion induction in the presence of protein synthesis inhibitors.

Retention of transmigrated cells in the subendothelium might be influenced by their interaction with SMCs. We found that E-LDL indeed promoted expression of ICAM-1 in these cells, which in turn enhanced the adhesion predominantly of T lymphocytes. The expression of ICAM-1 by human SMCs has been observed after stimulation with TNF-{alpha}.51 52

These collective findings would be in accordance with the concept that E-LDL may stimulate ECs to selectively recruit monocytes and T-cells at an early stage of fatty streak formation. Once large numbers of macrophages and T-cells are present, there is a high potential for further enzymatic modification of LDL. Together with chemotactic factors generated by complement activation and by macrophage-foam cells, E-LDL may sustain monocyte and T-cell migration into the vessel wall, thus promoting lesion progression. Thereby, monocyte migration into the subendothelium may also perpetuate atherogenesis, inasmuch as enhanced insudation of native LDL across EC barriers appears to accompany cell transmigration.53 Once having entered the vessel wall, inflammatory cells may be retained by interactions with proteoglycans54 55 and with adhesion molecules expressed on tissue cells. In this regard, our observation that ICAM-1 is upregulated on SMCs on exposure to E-LDL could be of relevance.

Overall, this study thus goes further to identify E-LDL as a potentially relevant initiator of atherosclerosis, rivaling ox-LDL in its potency to stimulate adhesion and transmigration of monocytes and T lymphocytes through EC monolayers. Inasmuch as the molecular mechanisms underlying these processes require further investigations, the present findings are relevant at this stage because they underscore the fact that LDL need not necessarily be modified by oxidation to acquire important atherogenic properties.


*    Acknowledgments
 
We are very grateful to Prof. Dr. W. Schmiedt, Department of Thoracic Surgery (University Mainz), for providing us with aortic specimens and the staff of the Vincenz and Elisabethen-Hospital, Mainz, for providing us with umbilical cords. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 311, grant D10 to S.B. and in part by grant Pr 372/1-3 to K.T.P.) and the Verband der Chemischen Industrie.

Received June 17, 1998; accepted October 21, 1998.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
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 *References
 

  1. Stary HC. Macrophages, macrophage foam cells, and eccentric intimal thickening in the coronary arteries of young children. Atherosclerosis. 1987;64:91–108.[Medline] [Order article via Infotrieve]
  2. Gerrity RG. The role of the monocyte in atherogenesis I: transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol. 1981;103:181–190.[Abstract]
  3. Gerrity RG. The role of the monocyte in atherogenesis II: migration of foam cells from atherosclerotic lesions. Am J Pathol. 1981;103:191200.
  4. Rosenfeld ME, Tsukada T, Chait A, Bierman EL, Gown AM, Ross R. Fatty streak expansion and maturation in Watanabe Heritable Hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1987;7:24–34.[Abstract]
  5. Watanabe T, Hirata M, Yoshikawa Y, Nagafuchi Y, Toyoshima H, Watanabe T. Role of macrophages in atherosclerosis: sequential observations of cholesterol-induced rabbit aortic lesion by the immunoperoxidase technique using monoclonal antimacrophage antibody. Lab Invest. 1985;53:80–90.[Medline] [Order article via Infotrieve]
  6. Rosenfeld ME, Tsukada T, Gown AM, Ross R. Fatty streak initiation in Watanabe Heritable Hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1987;7:9–23.[Abstract]
  7. Emeson EE, Robertson AL Jr. T lymphocytes in aortic and coronary intimas: their potential role in atherogenesis. Am J Pathol. 1988;130:369–376.[Abstract]
  8. van der Wal AC, Das PK, Bentz van de Berg D, van der Loos CM, Becker AE. Atherosclerotic lesions in humans: in situ immunophenotypic analysis suggesting an immune mediated response. Lab Invest 1989; 61:166–170.
  9. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol. 1989;135:169–175.[Abstract]
  10. Kishikawa H, Shimokama T, Watanabe T. Localization of T lymphocytes and macrophages expressing IL-1, IL-2 receptor, IL-6 and TNF in human aortic intima: role of cell-mediated immunity in human atherogenesis. Virchows Arch A Pathol Anat Histopathol. 1993;423:433–442.[Medline] [Order article via Infotrieve]
  11. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T-cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986;6:131–138.[Abstract/Free Full Text]
  12. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines. Adv Immunol. 1994;55:97–179.[Medline] [Order article via Infotrieve]
  13. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76:301–314.[Medline] [Order article via Infotrieve]
  14. Baggiolini M, Dewald B, Moser B. Human chemokines: an update. Annu Rev Immunol. 1997;15:675–705.[Medline] [Order article via Infotrieve]
  15. Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol. 1995;57:827–872.[Medline] [Order article via Infotrieve]
  16. Okada M, Sugita O, Miida T, Matsuto T, Inano K. Effects of modified low density lipoprotein and hypoxia on the expression of endothelial leukocyte adhesion molecule-1. Presse Med. 1995;24:483–488.
  17. Mehta A, Yang B, Khan S, Hendricks JB, Stephen C, Mehta JL. Oxidized low-density lipoproteins facilitate leukocyte adhesion to aortic intima without affecting endothelium-dependent relaxation: role of P-selectin. Arterioscler Thromb Vasc Biol. 1995;15:2076–2083.[Abstract/Free Full Text]
  18. Jeng JR, Chang CH, Shieh SM, Chiu HC. Oxidized low-density lipoprotein enhances monocyte-endothelial cell binding against shear-stress-induced detachment. Biochim Biophys Acta. 1993;1178:221–227.[Medline] [Order article via Infotrieve]
  19. Territo MC, Berliner JA, Almada L, Ramirez R, Fogelman AM. Beta-very low density lipoprotein pretreatment of endothelial monolayers increases monocyte adhesion. Arteriosclerosis. 1989;9:824–828.[Abstract/Free Full Text]
  20. Endemann G, Pronzcuk A, Friedman G, Lindsey S, Alderson L, Hayes KC. Monocyte adherence to endothelial cells in vitro is increased by beta-VLDL. Am J Pathol. 1987;126:1–6.[Abstract]
  21. Weber C, Erl W, Pietsch A, Strobel M, Ziegler-Heitbrock HW, Weber PC. Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. Arterioscler Thromb. 1994;14:1665–1673.[Abstract/Free Full Text]
  22. Weber C, Erl W, Weber PC. Enhancement of monocyte adhesion to endothelial cells by oxidatively modified low-density lipoprotein is mediated by activation of CD11b. Biochem Biophys Res Commun. 1995;206:621–628.[Medline] [Order article via Infotrieve]
  23. Kume N, Cybulsky MI, Gimbrone MA. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90:1138–1144.
  24. Schwartz D, Andalibi A, Chaverri-Almada L, Berliner JA, Kirchgessner T, Fang ZT, Tekamp-Olson P, Lusis AJ, Gallegos C, Fogelman AM, et al. Role of the GRO family of chemokines in monocyte adhesion to MM-LDL-stimulated endothelium. J Clin Invest. 1994;94:1968–1973.
  25. Navab M, Hough GP, Stevenson LW, Drinkwater DC, Laks H, Fogelman AM. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells. J Clin Invest. 1988;82:1853–1863.
  26. Navab M, Hama SY, Van Lenten BJ, Drinkwater DC, Laks H, Fogelman AM. A new antiinflammatory compound, leumedin, inhibits modification of low density lipoprotein and the resulting monocyte transmigration into the subendothelial space of cocultures of human aortic wall cells. J Clin Invest. 1993;91:1225–1230.
  27. Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA, Drinkwater DC, Laks H, et al. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest. 1991;88:2039–2046.
  28. Bhakdi S, Dorweiler B, Kirchmann R, Torzewski J, Weise E, Tranum-Jensen J, Walev I, Wieland E. On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety. J Exp Med. 1995;182:1959–1971.[Abstract/Free Full Text]
  29. Klouche M, Gottschling S, Gerl V, Hell W, Husmann M, Dorweiler B, Messner M, Bhakdi S. Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages. Arterioscler Thromb Vasc Biol. 1998;18:1376–1385.[Abstract/Free Full Text]
  30. Seifert PS, Hugo F, Tranum-Jensen J, Zahringer U, Muhly M, Bhakdi S. Isolation and characterization of a complement-activating lipid extracted from human atherosclerotic lesions. J Exp Med. 1990;172:547–557.[Abstract/Free Full Text]
  31. Seifert PS, Hugo F, Hansson GK, Bhakdi S. Prelesional complement activation in experimental atherosclerosis: terminal C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits. Lab Invest. 1989;60:747–754.[Medline] [Order article via Infotrieve]
  32. Vlaicu R, Niculescu F, Rus HG, Cristea A. Immunohistochemical localization of the terminal C5b-9 complement complex in human aortic fibrous plaque. Atherosclerosis. 1985;57:163–177.[Medline] [Order article via Infotrieve]
  33. Schmiedt W, Kinscherf R, Deigner HP, Kamencic H, Nauen O, Kilo J, Oelert H, Metz J, Bhakdi S. Complement C6 deficiency protects against diet-induced atherosclerosis in rabbits. Arterioscler Thromb Vasc Biol. 1998;18:1790–1795.[Abstract/Free Full Text]
  34. Torzewski M, Klouche M, Hock J, Messner M, Dorweiler B, Torzewski J, Gabbert HE, Bhakdi S. Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion. Arterioscler Thromb Vasc Biol. 1998;18:369–378.[Abstract/Free Full Text]
  35. Ross R. The smooth muscle cell II: growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol. 1971;50:172–186.[Abstract/Free Full Text]
  36. Magnier C, Papp B, Corvazier E, Bredoux R, Wuytack F, Eggermont J, Maclouf J, Enouf J. Regulation of sarco-endoplasmic reticulum Ca(2+)-ATPases during platelet-derived growth factor-induced smooth muscle cell proliferation. J Biol Chem. 1992;267:15808–15815.[Abstract/Free Full Text]
  37. Pawlowski NA, Kaplan G, Hamill AL, Cohn ZA, Scott WA. Arachidonic acid metabolism by human monocytes: studies with platelet-depleted cultures. J Exp Med. 1983;158:393–412.[Abstract/Free Full Text]
  38. Lindgren FT, Adamson GL, Jenson LC, Wood PD. Lipid and lipoprotein measurements in a normal adult American population. Lipids. 1975;10:750–756.[Medline] [Order article via Infotrieve]
  39. Wieland E, Parthasarathy S, Steinberg D. Peroxidase-dependent metal-independent oxidation of low density lipoprotein in vitro: a model for in vivo oxidation? Proc Natl Acad Sci U S A. 1993;90:5929–5933.[Abstract/Free Full Text]
  40. Taylor RF, Price TH, Schwartz SM, Dale DC. Neutrophil-endothelial cell interactions on endothelial monolayers grown on micropore filters. J Clin Invest. 1981;67:584–587.
  41. Munro JM, Cotran RS. The pathogenesis of atherosclerosis: atherogenesis and inflammation. Lab Invest. 1988;58:249–261.[Medline] [Order article via Infotrieve]
  42. Shimokama T, Haraoka S, Watanabe T. Immunohistochemical and ultrastructural demonstration of the lymphocyte-macrophage interaction in human aortic intima. Mod Pathol. 1991;4:101–107.[Medline] [Order article via Infotrieve]
  43. Shimokama T, Haraoka S, Watanabe T. Participation of T lymphocytes and macrophages in atherogenesis. J Atheroscler Thromb. 1994;1(suppl 1):S14–S20.
  44. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990;85:1260–1266.
  45. Faggiotto A, Ross R, Harker L. Studies of hypercholesterolemia in the nonhuman primate I: changes that lead to fatty streak formation. Arteriosclerosis. 1984;4:323–340.[Abstract/Free Full Text]
  46. Poston RN, Haskard DO, Coucher JR, Gall NP, Johnson Tidey RR. Expression of intercellular adhesion molecule-1 in atherosclerotic plaques. Am J Pathol. 1992;140:665–673.[Abstract]
  47. Duplaa C, Couffinhal T, Labat L, Moreau C, Petit-Jean ME, Doutre MS, Lamaziere JM, Bonnet J. Monocyte/macrophage recruitment and expression of endothelial adhesion proteins in human atherosclerotic lesions. Atherosclerosis. 1996;121:253–266.[Medline] [Order article via Infotrieve]
  48. Blann AD, McCollum CN. Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis. Thromb Haemost. 1994;72:151–154.[Medline] [Order article via Infotrieve]
  49. Rival Y, Del Maschio A, Rabiet MJ, Dejana E, Duperray A. Inhibition of platelet endothelial cell adhesion molecule-1 synthesis and leukocyte transmigration in endothelial cells by the combined action of TNF-alpha and IFN-gamma. J Immunol. 1996;157:1233–1241.[Abstract]
  50. Richardson M, Hadcock SJ, DeReske M, Cybulsky MI. Increased expression in vivo of VCAM-1 and E-selectin by the aortic endothelium of normolipemic and hyperlipemic diabetic rabbits. Arterioscler Thromb. 1994;14:760–769.[Abstract/Free Full Text]
  51. Couffinhal T, Duplaa C, Moreau C, Lamaziere JM, Bonnet J. Regulation of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human vascular smooth muscle cells. Circ Res. 1994;74:225–234.[Abstract/Free Full Text]
  52. Couffinhal T, Duplaa C, Labat L, Lamaziere JM, Moreau C, Printseva O, Bonnet J. Tumor necrosis factor-alpha stimulates ICAM-1 expression in human vascular smooth muscle cells. Arterioscler Thromb. 1993;13:407–414.[Abstract/Free Full Text]
  53. Territo M, Berliner JA, Fogelman AM. Effect of monocyte migration on low density lipoprotein transport across aortic endothelial cell monolayers. J Clin Invest. 1984;74:2279–2284.
  54. Mahley RW, Weisgraber KH, Innerarity TL. Interaction of plasma lipoproteins containing apolipoproteins B and E with heparin and cell surface receptors. Biochim Biophys Acta. 1979;575:81–91.[Medline] [Order article via Infotrieve]
  55. Hirose N, Blankenship DT, Krivanek MA, Jackson RL, Cardin AD. Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B. Biochemistry. 1987;26:5505–5512.[Medline] [Order article via Infotrieve]



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