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(Circulation. 1998;98:2255-2261.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

-Tocopherol Enrichment of Monocytes Decreases Agonist-Induced Adhesion to Human Endothelial Cells

Kazi Nazrul Islam, PhD; Sridevi Devaraj, PhD; ; Ishwarlal Jialal, MD, PhD

From the Departments of Pathology (K.N.I., S.D., I.J.) and Internal Medicine (I.J.), University of Texas Southwestern Medical Center at Dallas.

Correspondence to Ishwarlal Jialal, MD, PhD, Department of Pathology and Internal Medicine, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235-9073. E-mail jialal.i{at}pathology.swmed.edu

	Abstract

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Background—Monocyte-endothelium adhesion is a crucial early event in atherogenesis. Several reports indicate that -tocopherol (AT) is a potent antioxidant in plasma and LDL and also has intracellular effects that are antiatherogenic. Recently, it has been shown that AT supplementation results in decreased monocyte–endothelial cell adhesion. However, there is a paucity of data on the mechanisms by which AT inhibits adhesion of monocytes. We studied the effect of AT enrichment of a human monocytic cell line, U937, on adhesion to human umbilical vein endothelial cells (HUVECs).

Methods and Results—Both lipopolysaccharide (LPS)– and N-formyl-methionyl-leucyl-phenylalanine (FMLP)–stimulated U937 adhesion to HUVECs were studied. AT (50 and 100 µmol/L) significantly decreased adhesion of both LPS- and FMLP-stimulated U937 cells to HUVECs (LPS-treated cells, P<0.0125; FMLP-treated cells, P<0.05). Expression of the adhesion molecules CD11a, CD11b, CD11c, very late antigen-4 (VLA-4), and L-selectin, as assessed by flow cytometry, was increased in the stimulated U937 cells, and AT resulted in significant reduction in the expression of CD11b and VLA-4. In addition, activation of the transcription factor nuclear factor-B (NF-B), as assessed by gel shift assays, was inhibited by pretreatment with AT in LPS-treated U937 cells.

Conclusions—AT significantly decreases adhesion of activated monocytes to endothelial cells by decreasing expression of CD11b and VLA-4 on monocytes, possibly by inhibiting the activation of NF-B.

Key Words: cell adhesion molecules • antioxidants • endothelium • NF-kappa B

	Introduction

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The monocyte-macrophage is a critical cell in atherogenesis.^{1 2} An early event in the genesis of the fatty streak lesion is the attachment of monocytes to vascular endothelium. After monocytes have adhered to the endothelium, they migrate into the intima, imbibe lipid, and become foam cells. The exact mechanism by which monocyte-endothelium adhesion occurs in vivo remains to be elucidated. Recent work has identified specific adhesion molecules on endothelial cells (ECs) and monocytes.³ These adhesion molecules include E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) on ECs and members of the ß-2 integrin family (CD11a, CD11b, and CD11c/18) and the ß-1 integrin VLA-4 (very late antigen-4 [CD49d/29]) on monocytes. Modulation of monocyte-EC adhesion could be an important target in the treatment of atherosclerosis. It has also been shown that agonist-induced adhesion of monocytes to the endothelium is mediated by activation of the transcription factor nuclear factor-B (NF-B).⁴ Recent findings implicate reactive oxygen species in NF-B activation.⁵ Thus, the modulation of these processes, ie, activation of NF-B, expression of adhesion molecules, and monocyte-EC adhesion by antioxidants, assumes great significance. Recently, Weber et al⁶ showed that increased adhesion of monocytes to ECs in smokers with increased expression of CD11b could be prevented by vitamin C supplementation.

-Tocopherol (AT) is a potent lipophilic antioxidant that protects membranes from lipid peroxidation.^{7 8} In addition to decreasing the oxidative susceptibility of LDL, AT has been shown to have direct antiatherogenic effects on cells.⁹ Recently, it has been shown that supplementation of human volunteers with AT (1200 IU/d) significantly decreased adhesion of human monocytes to human umbilical vein endothelial cells (HUVECs) and decreased the secretion of interleukin-1ß.¹⁰ Faruqi et al¹¹ showed that AT enrichment of ECs inhibited agonist-induced adhesion of U937 cells to HUVECs. More recently, Martin et al¹² showed that in vitro enrichment of human aortic ECs with AT significantly inhibited LDL-induced adhesion of monocytes to ECs in a dose-dependent manner, with a concomitant reduction in levels of soluble ICAM-1. However, to date there are no studies examining the effect of AT enrichment of monocytes on adhesion. Thus, in the present study, we examined the effect of AT enrichment of monocytes on subsequent adhesion to ECs after activation with 2 agonists, lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). The effect of AT on the expression of adhesion molecules on monocytes and on NF-B activation was also studied to gain insight into the mechanisms by which AT mediated this effect.

	Methods

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Reagents
The reagents used for the experiments were as follows: RPMI 1640, endothelial cell growth medium (EGM), antibiotics, leupeptin, PMSF, pyrrolidinedithiocarbamate (PDTC), DTT, EGTA, and Nonidet P-40 were from Sigma Chemical Co; 5- and 6-carboxyfluorescein diacetate succinimidyl ester [5(6)-CFDA SE] were from Molecular Probes Inc; poly (dI-dC) was from Pharmacia; FMLP was from Peninsula Laboratories; and the monoclonal antibodies anti-CD11a-FITC, anti-CD11b-PE, anti-CD11c-PE, VLA-4, and anti-L-selectin were from Becton Dickinson. Antibody to the p65 subunit of NF-B was purchased from Boehringer Mannheim.

Cell Cultures
HUVECs were purchased from Clonetics Laboratories and cultured in EGM as described previously.^{13 14} HUVECs were cultured in EGM and were maintained at confluence in 5% CO₂-95% air and passaged according to standard procedures. HUVECs were used 24 hours after confluence between 3 and 10 passages. Isolated ECs were tested by the vendor (Clonetics) to have an endotoxin concentration of <0.125 EU (endotoxin units)/mL. The human monocytic tumor cell line U937 was purchased from American Type Culture Collection and maintained at 0.5 to 1.0 million cells/mL in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS) containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine. Cells were split with fresh media 1:5 every 2 to 3 days. Cell counts were performed routinely to maintain low population density.

Supplementation of U937 Cells With -Tocopherol
For all experiments of monocyte-EC adhesion, expression of counterreceptors, and activation of the transcription factor NFB, 1 set of U937 cells was incubated overnight with the specified concentrations of AT as described in the figures and below and another set was incubated with DMSO as vehicle control. After overnight incubation, cells were pelleted, washed, and incubated again with DMSO or AT for 30 minutes at a similar concentration before activation with LPS or FMLP.

Monocyte-EC Adhesion
HUVECs were seeded in 24-well plates for 3 to 4 days before the experiment. Only confluent monolayers were used, as confirmed by microscopic inspection the day before the assay. U937 cells were incubated in the absence and presence of different concentrations of AT (25, 50, and 100 µmol/L) overnight in RPMI 1640 medium containing 10% FBS at 37°C. A stock solution of AT (10 mmol/L) was prepared in DMSO solution, and 10 µL was added to 1 mL of U937 cells to give a final concentration of 100 µmol/L AT in 1% DMSO. DMSO was added as vehicle control to cells that were not incubated with AT. Cells were pelleted and reconstituted in RPMI containing 2% FBS. After 20 to 30 minutes' preincubation with or without AT, 1 set of cells was incubated with 100 ng/mL LPS for 2 hours and another set with 10^-7 mol/L FMLP for 30 minutes at 37°C. After a defined incubation period as determined from preliminary experiments (2 hours for LPS or 30 minutes for FMLP), cells were again pelleted and reconstituted in RPMI containing 2% FBS and 4 µmol/L of the fluorescent dye 5(6)-CFDA SE.^{15 16} Before addition of dye, an aliquot of cells was kept for fluorescent-activated cell sorter (FACS) analysis of adhesion molecules. After 30 minutes' incubation at 37°C, dye loading was stopped by the addition of an excess of cold RPMI containing 2% FBS. Fluorescence-labeled cells were pelleted and resuspended (1x10⁶/mL) in EGM. HUVECs were washed twice with EGM before addition of fluorescence-loaded U937 cells (0.5x10⁶/mL) and incubated at 37°C, 5% CO₂ at 90% humidity. After 30 minutes, the cell supernatant was aspirated, and the bound cells were gently washed twice with phenol-free RPMI 1640. Cells were lysed with 1 mL of 0.01% Triton X-100 in 0.1 mol/L Tris buffer per well (pH 8.0), and fluorescence was measured with excitation and emission wavelengths of 485 and 535 nm, respectively. Unactivated U937 cells were used to assess basal adhesion. Wells containing HUVECs only were used as blanks. To simulate the in vivo supplemented state, both HUVECs and U937 cells were pretreated overnight with AT to determine if incubation of both types of cells with AT resulted in further inhibition of adhesion.

FACS Analysis of Adhesion Molecules
Expression of counterreceptors (CD11a/18, CD11b/18, CD11c/18, CD49d/18, and L-selectin) on LPS- and FMLP-activated monocytes was assessed by flow cytometry with a FACScan flow cytometer (Becton Dickinson). LPS- and FMLP-activated monocytes (1x10⁶ cells/mL), with or without preincubation with AT, were stained by routine methods with saturating amounts of directly conjugated, commercially available monoclonal antibodies with phycoerythrin (PE), FITC, and tricolor (TC) fluorochromes. To avoid nonspecific binding to Fc receptors, cells were preincubated with 5% human serum in PBS for 15 minutes on ice, washed, and then stained. Cells were fixed with 1% paraformaldehyde before being acquired by the FACScan flow cytometer. Isotype-matched PE, FITC, and TC controls were run with each sample. Samples were analyzed with "Paint-a-gate" software (Becton Dickinson) and results expressed in units of mean fluorescence intensity.

Electrophoretic Mobility Shift Assay
U937 cells were incubated with or without different concentrations of AT (25, 50, and 100 µmol/L) overnight in RPMI 1640 medium containing 10% FBS at 37°C as described above. Cells were pelleted and reconstituted in RPMI containing 10% FBS. After 30 minutes' additional preincubation in the absence or presence of AT, cells were activated with 100 ng/mL LPS for 2 hours at 37°C. Preliminary dose-response studies indicated that the optimum dose of LPS that activated NF-B was 100 ng/mL. This is in accord with the published literature.^{11 12 17} Because PDTC is known to inhibit NF-B activation, cells were also preincubated with PDTC (100 µmol/L) before activation with LPS. Nuclear extracts were prepared from 1x10⁶ treated cells according to Staal et al¹⁸ with some modification. Briefly, cells were harvested, centrifuged for 5 minutes at 14 000 rpm, and washed in 1 mL of ice-cold PBS. Cells were pelleted, resuspended in 0.2 mL of buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 0.1 mmol/L EDTA; 0.1 mmol/L EGTA; 2.5 mmol/L DTT; 1 mmol/L PMSF; and 5 µg/mL leupeptin) and incubated on ice for 15 minutes. After addition of a 10% Nonidet P-40 solution (12.5 µL), cells were vigorously mixed for 10 seconds and centrifuged for 60 seconds at 14 000 rpm at 4°C. Pelleted nuclei were resuspended in 50 µL of buffer C (20 mmol/L HEPES, pH 7.9; 25% [vol/vol] glycerol; 0.4 mol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA; 1 mmol/L PMSF; and 5 µg/mL leupeptin), mixed for 10 minutes, and centrifuged for 5 minutes at 14 000 rpm at 4°C. The supernatant containing the nuclear proteins was harvested, protein concentration was determined by Bio-Rad Protein assay, and the supernatant was stored at -80°C until use.

Electrophoretic mobility shift assay (EMSA) was performed essentially as described previously.^{19 20} Binding reaction mixtures (20 µL) containing 5 µg of protein of the nuclear extract, 2 µg of poly (dI-dC), 50 mmol/L NaCl, 4% glycerol, 2.5 mmol/L EDTA, 2.5 mmol/L DTT, 5 mmol/L MgCl₂, ³²P-labeled probe, and 10 mmol/L Tris-HCl (pH 7.5) were incubated for 30 minutes at room temperature. A double-stranded NF-B oligonucleotide probe containing HIV-B enhancer DNA template with a tandem duplicate of an NF-B binding site (GGGACTTTCC) was used. Incubation was also conducted in the presence of nonspecific competitor DNA (TTTACTTTCC). Products were separated by electrophoresis through a native 4% polyacrylamide gel in a running buffer of 12.5 mmol/L Tris-borate (pH 8.0) containing 0.25 mmol/L EDTA at 150 V for 2.5 hours, and dried gels were analyzed by autoradiography. Control reactions with 250-fold molar excess of unlabeled wild-type and mutant oligonucleotide probes were performed to demonstrate the specificity of the shifted DNA-protein complexes for NF-B. In addition, supershift experiments were conducted in the presence of p65 antibody (2 µg) to demonstrate correspondence of the band to NF-B/Rel transcription factor.

Statistical Analysis
Results are expressed as mean±SD. Repeated-measures ANOVA was performed to assess overall differences between the different treatments, as described previously.¹⁰ Fisher's least significant difference method was applied for multiple comparisons. Paired t tests were used to compare the differences between treatments.

	Results

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There was a significant increase in AT content in U937 cells after pretreatment for 24 hours with 100 µmol/L AT (baseline: 0.09±0.04 versus 2.11±0.99 nmol/mg cell protein in AT-enriched cells; n=4 experiments; P<0.001). As can be seen in Figure 1A, LPS (100 ng/mL)-induced activation of U937 cells significantly increased adhesion to HUVECs compared with DMSO-treated control (P=0.0029). Repeated-measures ANOVA showed that pretreatment of U937 cells with AT before activation with LPS or FMLP affected adhesion to HUVECs (P<0.001). Pretreatment of U937 cells with 50 or 100 µmol/L AT before activation with LPS decreased adhesion of U937 to HUVECs (LPS, 5.7±2.0 fluorescence units; LPS plus AT 50 µmol/L, 4.5±2.8 fluorescence units; LPS plus AT 100 µmol/L, 3.8±1.3 fluorescence units; P<0.0125 compared with LPS alone). To determine whether the inhibitory effect of AT was specific for LPS-induced adhesion, we tested the effect of AT on another agonist, the chemotactic peptide FMLP (Figure 1B). FMLP also significantly increased adhesion of U937 cells to HUVECs (P=0.0151). AT significantly inhibited this increased adhesion (FMLP, 10.3±3.6 fluorescence units; FMLP plus AT 50 µmol/L, 8.4±2.8 fluorescence units; FMLP plus AT 100 µmol/L, 7.6±2.8 fluorescence units; P<0.05 compared with FMLP alone).

View larger version (22K): [in this window] [in a new window]   Figure 1. Agonist-induced adhesion of U937 cells to HUVECs. Treated cells (with or without AT) were incubated with 100 ng/mL of LPS for 2 hours (A) and with 10-7 mol/L FMLP for 30 minutes (B) at 37°C as described in Methods. Cells were labeled with 4 µm of the fluorescence dye CFDA for 30 minutes, and labeled cells (0.5x106/mL) were added to HUVECs and incubated for 30 minutes at 37°C. After washing, cells were lysed with 1 mL of 0.01% Triton X-100 in 0.1 mol/L Tris buffer per well (pH 8.0), and fluorescence was measured with excitation and emission wavelengths of 485 and 535 nm, respectively. Results are presented as mean±SD of 4 experiments. A: *P<0.005 compared with control, **P<0.0125 compared with LPS. B: *P<0.02 compared with control, **P<0.05 compared with FMLP.

In addition, pretreatment of both activated U937 cells with AT (100 µmol/L) and HUVECs with AT (50 µmol/L) resulted in additional inhibition of monocyte-EC adhesion compared with LPS-activated cells (U937 cells plus AT 100 µmol/L, 49% inhibition; U937 plus AT 100 µmol/L and ECs plus AT 50 µmol/L, 62% inhibition; P<0.05; n=3 experiments).

The effect of AT enrichment of U937 cells on the expression of adhesion molecules on activated monocytes as determined by flow cytometry is shown in Figure 2. After stimulation with either FMLP or LPS, there was a significant increase in the expression of CD11a, CD11b, CD11c, VLA-4 and L-selectin compared with unstimulated cells (P<0.02). There was no significant change in L-selectin after activation of U937 cells with LPS or FMLP. Repeated-measures ANOVA showed that pretreatment of U937 cells with AT before stimulation with LPS or FMLP affected the expression of adhesion molecules (P<0.0005). AT (100 µmol/L) resulted in a significant reduction in the expression of CD11b and VLA-4 in both LPS- and FMLP-stimulated cells (percent reduction by AT in LPS-activated cells: CD11b, 18%; VLA-4, 17%; P<0.01; percent reduction by AT in FMLP-stimulated cells: CD11b, 15%; VLA-4, 15.3%; P<0.01). There was no significant decrease in CD11a, CD11c, or L-selectin expression with AT.

View larger version (34K): [in this window] [in a new window]   Figure 2. Effect of AT enrichment of monocytes on the expression of adhesion molecules from activated monocytes. After stimulation of U937 cells with either FMLP (10-7 mol/L) or LPS (100 ng/mL), expression of adhesion molecules CD11a, CD11b, CD11c, VLA-4, and L-selectin was measured by flow cytometry in cells incubated in the presence and absence of AT (100 µmol/L). Values are mean of 5 experiments done in duplicate and expressed as mean fluorescence units ±SD. *P<0.01 compared with LPS- or FMLP-activated cells.

The increase in the agonist-induced adhesion of U937 cells to HUVECs might be caused by the activation of NF-B in U937 cells. Therefore, gel shift mobility assays for the treated U937 cells were performed. Cells were incubated with or without AT and PDTC and then activated with LPS (100 ng/mL). Nuclear extracts were prepared from the treated cells and analyzed for the specific DNA binding of NF-B with EMSAs. As shown in lane 3 in Figure 3, LPS activated the transcription factor NF-B. This was inhibited by AT (50 and 100 µmol/L) and PDTC, a known inhibitor of NF-B activation. To establish the specificity of the shifted bands, competition analysis was performed with nuclear extracts from cells incubated with 250-fold excess of unlabeled wild-type and mutant NF-B probes; Although the cold mutant probe had no effect, incubation with excess cold wild-type probe resulted in complete elimination of the radioactive NF-B signal (Figure 4A). Incubation of LPS-activated nuclear extracts with p65 antibody induced a supershifting of the band, demonstrating its correspondence to the NF-B/Rel transcription factor (Figure 4B). In addition, when radiolabeled mutant probe was used instead of radiolabeled wildtype, there was a 10-fold decrease in NF-B expression in LPS-activated cells (data not shown).

View larger version (65K): [in this window] [in a new window]   Figure 3. Effects of AT on activation of NF-B in LPS-stimulated U937 cells. Nuclear extracts were prepared from cells treated with DMSO (lane 2); LPS (lane 3); LPS plus 25, 50, and 100 µmol/L AT (lanes 4 to 6, respectively); and LPS plus PDTC (lane 7), followed by EMSA as described in Methods. Free probe is shown in lane 1.

View larger version (37K): [in this window] [in a new window]   Figure 4. A, Effect of unlabeled wild-type and mutant probes on activation of NF-B in LPS-stimulated U937 cells. Nuclear extracts were prepared from cells treated with LPS followed by EMSA as described in Methods. Lane 1 represents LPS; lane 2, LPS plus 250-molar excess cold mutant probe; and lane 3, LPS plus 250-molar excess cold wild-type probe. B, Supershift of NF-B in LPS-stimulated U937 cells. Nuclear extracts were prepared from cells treated with LPS followed by EMSA as described in Methods. Lane 1 represents LPS; lane 2, LPS plus p65 antibody (2 µg).

	Discussion

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There are few data on the effect of antioxidants such as AT on monocyte-EC adhesion. Faruqi et al¹¹ observed that when ECs were pretreated with AT before stimulation with agonists (phorbol ester, interleukin-1, thrombin, and LPS), there was less agonist-induced adhesion of monocytes to ECs. Although Faruqi et al did not show a decrease in NF-B with E-selectin consensus sequence in the gel shift assay, they demonstrated a decrease in E-selectin mRNA and cell surface expression. Recently, Martin et al¹² showed that in vitro enrichment of ECs with AT significantly inhibited LDL-induced adhesion of monocytes to ECs in a dose-dependent manner and decreased soluble ICAM-1 release. They did not study the effect of AT on NF-B activity. Although these studies suggest that AT enrichment of ECs decreases adhesion of monocytes to endothelium, there are no studies to date that examine the effect of AT enrichment of monocytes on monocyte-EC adhesion. In a recent study¹⁰ that tested the effect of AT supplementation (1200 IU/d) on monocyte function, we showed that AT significantly decreases adhesion of human monocytes to endothelium. Like Faruqi et al,¹¹ we could not explain this via an inhibition of protein kinase C by AT. The present study was undertaken to elucidate other potential mechanisms.

In the present study, we have demonstrated that preincubation of U937 monocytic cells with AT resulted in significant enrichment of monocytes with AT. In addition, at doses between 25 and 100 µmol/L, AT was not cytotoxic. Furthermore, after enrichment, there was significant inhibition of both LPS- and FMLP-induced U937 cell adhesion to HUVECs at doses 50 µmol/L AT. These levels can be attained in plasma after AT supplementation in vivo.^{21 22} Faruqi et al¹¹ observed in ECs that AT inhibited agonist-induced monocyte adhesion to ECs, with an IC₅₀ of 45 µmol/L. Martin et al¹² also reported a significant reduction in LDL-induced monocyte-EC adhesion after preincubation of ECs with 42 µmol/L AT. Because we have shown that AT enrichment of monocytes decreases adhesion to endothelium, and previous workers have shown that AT enrichment of HUVECs decreases adhesion, one can speculate that AT supplementation in vivo will result in a greater inhibition of monocyte-EC adhesion because AT will partition in both cells. We confirmed this by demonstrating that preincubation of both U937 cells and HUVECs with AT results in a greater inhibition of adhesion than preincubation of U937 cells alone.

To delineate mechanisms through which AT may be acting to reduce adhesion, we studied the effect of AT on the expression of adhesion molecules on monocytes and the role of AT in the activation of NF-B. Adhesion of monocytes to ECs is mediated by integrins that bind to counterreceptors on ECs. The most important counterreceptors expressed on monocytes include CD11a/18 (LFA-1), CD11b/18 (Mac-1), and CD49d/29 (VLA-4), which bind to ICAM-1 and ICAM-2, ICAM-1, and VCAM-1 on the ECs, respectively. VCAM-1 and ICAM-1 expression has been demonstrated in human atherosclerotic lesions.^{23 24 25 26}

In the present study, we have shown that the expression of CD11a, CD11b, CD11c, VLA-4, and L-selectin on U937 cells is increased after stimulation with LPS or FMLP. In addition, preincubation of U937 cells with AT significantly decreased the expression of CD11b and VLA-4 from these cells. Weber et al⁶ showed that long-term cigarette smoking was associated with a CD11b-dependent increase in adhesiveness of isolated human monocytes to endothelium, which was decreased after supplementation with ascorbate for 10 days. In previous studies, Weber et al²⁷ showed that enhancement of monocyte adhesion to ECs by modified LDL was mediated via activation of CD11b. They also showed that enhancement of adhesion was prevented by an anti-CD11b monoclonal antibody. In addition, Ragab et al²⁸ reported that oxidized lipoprotein(a) and LDL increased CD11b expression by 60% in U937 cells, with a concomitant increase in the adhesion of U937 cells to cultured ECs. Martin et al¹² demonstrated that vitamin E inhibits LDL-induced monocyte-EC adhesion in part by decreasing expression of soluble ICAM-1, which is a counterreceptor for CD11b. Although there are few studies on the role of VLA-4 in monocyte-EC adhesion, Cominacini et al²⁹ showed that pretreatment of HUVECs or LDL with antioxidants such as AT and probucol significantly reduced the expression of both ICAM-1 and VCAM-1 on HUVECs induced by oxidized LDL. In addition, Marui et al³⁰ reported that VCAM-1 gene transcription and expression in human vascular ECs are increased in the presence of interleukin-1ß, LPS, and tumor necrosis factor- (TNF) and can be repressed by 90% by the antioxidants PDTC and N-acetyl cysteine. This is supported by the recent finding that the ability of ECs to express VCAM-1 in response to cytokine stimulation may be modulated by oxidized LDL by a redox-sensitive mode of regulation.³¹ These studies suggest that oxidative stress is an important regulatory signal in the pathogenesis of atherosclerosis.

NF-B is a mammalian transcription factor that is directly involved in the activation of genes responsible for inflammation.³² In cells that have inducible NF-B activity, the factor comprises a p50-p65 heterodimer bound by an inhibitory subunit I-B in the cytosol and can be activated by dissociation of I-B.^{33 34} The importance of activated NF-B in atherosclerosis has been demonstrated by its presence in smooth muscle cells, macrophages, and ECs of human atherosclerotic lesion tissue but not in normal vessels.^{35 36} A variety of genes are induced in the atherosclerotic lesion that have been shown to be regulated by NF-B, including genes encoding TNF, interleukin-1, tissue factor, macrophage colony stimulating factor, VCAM-1, and ICAM-1. Schreck et al¹⁷ have suggested a novel signal transduction pathway for NF-B activation that involves reactive oxygen species as second messengers based on studies with Jurkat T cells that responded to the addition of exogenous hydrogen peroxide. Some of the antioxidants that have been shown to inhibit NF-B activation include PDTC, N-acetyl cysteine, and -lipoate.^{17 37 38} Suzuki and Packer³⁹ have shown that NF-B activation can be inhibited in TNF-–activated Jurkat T cells by treatment with vitamin E acetate and succinate, resulting in decreased DNA binding activity. However, they did not observe any inhibition with AT alone.

In the present study, we showed that pretreatment of U937 cells with AT significantly decreased the LPS-induced activation of NF-B. PDTC was used as a positive control in all experiments and has been shown by numerous investigators to effectively inhibit activation of NF-B. There have been conflicting reports on the effect of AT in NF-B activation. Whereas Suzuki and Packer³⁹ as well as Faruqi et al¹¹ did not show any effect of AT on activation of NF-B by pretreatment of Jurkat T cells and ECs, respectively, Hennig et al⁴⁰ showed that pretreatment of ECs for 24 hours with AT or 6 hours with N-acetyl cysteine significantly inhibited linoleic acid–induced activation of NF-B. It is possible that AT has differential effects in different cell lines. In addition, none of these investigators studied the effect of AT enrichment of monocytes on subsequent NF-B activation. This is clearly important, because the monocyte is a pivotal cell in atherogenesis, and its respiratory burst has been shown to lead to activation of NF-B.⁴¹

In the present study, we have shown that enrichment of monocytes with AT resulted in a significant decrease in adhesion of monocytes to ECs, mediated by a concomitant reduction in the expression of the adhesion molecules CD11b and VLA-4. This inhibition is possibly mediated by inhibition of the activation of NF-B. Support for this hypothesis comes from studies by Sokoloski et al,⁴² who showed that antisense oligonucleotides to the p65 subunit of NF-B block CD11b expression on the surface of FMLP- or phorbol ester–treated HL-60 human promyelocytic leukemia cells. However, additional studies in monocytes are required to confirm this.

In conclusion, these studies provide evidence for the beneficial effects of AT on a crucial early event in atherosclerosis, viz, monocyte-EC adhesion. This beneficial effect of AT further strengthens its evolving role as an adjunctive therapy in the management of atherosclerosis because in a recent study, although patients with dyslipidemia had elevated levels of soluble adhesion molecules, aggressive lipid-lowering therapy had only limited effects on their levels.⁴³ These and other studies support the concept that the possible beneficial effects of AT supplementation in reducing coronary artery disease can be attributed to its combined effects on inhibition of the oxidative modification of lipoproteins and its intracellular effects on cells critical in atherogenesis, such as monocytes.⁹

	Acknowledgments

 
The authors are thankful to Richard Scheuermann, PhD, Associate Professor, Department of Pathology, and his fellow, Zhiyong Wang, PhD, for providing the EMSA probe and allowing them to conduct the EMSA studies in his laboratory.

Received May 19, 1998; revision received July 14, 1998; accepted July 21, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
   
2. Libby P, Hanson GK. Biology of disease: involvement of the immune system in atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5–13.[Medline] [Order article via Infotrieve]
   
3. Bevilacqua MP, Nelson RM, Mannori G, Cecconi O. Endothelial-leukocyte adhesion molecules in human disease. Ann Rev Med. 1994;45:361–378.[Medline] [Order article via Infotrieve]
   
4. Baeuerle PA, Baltimore D. A 65KD subunit of NFb is required for inhibition of NF-b by IKb. Genes Dev. 1989;3:1689–1698.[Abstract/Free Full Text]
   
5. Ruben SM, Dillon PJ, Schreck R, Henkel T, Chen CH, Maher M, Baeuerle PA, Rosen C. Isolation of a rel-related human cDNA that encodes the 65-kD subunit of NF-b. Science. 1991;251:1490–1493.[Abstract/Free Full Text]
   
6. Weber C, Erl W, Weber K, Weber PC. Increased adhesiveness of isolated monocytes to endothelium is prevented by vitamin C intake in smokers. Circulation. 1996;93:1488–1492.[Abstract/Free Full Text]
   
7. Traber MG, Packer L. Vitamin E: beyond antioxidant function. Am J Clin Nutr. 1995;62:1501S–1509S.[Abstract/Free Full Text]
   
8. Packer L. Protective role of vitamin E in biological systems. Am J Clin Nutr. 1991;53:1050S–1055S.[Abstract/Free Full Text]
   
9. Devaraj S, Jialal I. The effects of AT on critical cells in atherogenesis. Curr Opin Lipidol. 1998;9:11–15.[Medline] [Order article via Infotrieve]
   
10. Devaraj S, Li D, Jialal I. The effects of AT supplementation on monocyte function-decreased lipid oxidation, IL-1ß secretion and monocyte adhesion to endothelium. J Clin Invest. 1996;98:756–763.[Medline] [Order article via Infotrieve]
    
11. Faruqi R, Motte C, Dicorleto PE. AT inhibits agonist-induced monocyte cell adhesion to cultured human endothelial cells. J Clin Invest. 1994;94:592–600.
    
12. Martin A, Foxall T, Blumberg JB, Meydani M. AT inhibits LDL-induced adhesion of monocytes to human aortic endothelial cells in vitro. Arterioscler Thromb Vasc Biol. 1997;17:429–436.[Abstract/Free Full Text]
    
13. Pritchard KA Jr, Wong PY-K, Stemerman MB. Atherogenic concentrations of LDL enhance endothelial cell generation of epoxyeicosatrienoic acid products. Am J Pathol. 1990;136:1381–1391.
    
14. Holland JA, Pritchard KA Jr, Rogers NJ, Stemerman MB. Perturbation of cultured human endothelial cells by atherogenic levels of LDL. Am J Pathol. 1988;132:474–478.[Abstract]
    
15. Smalley DM, Lin JHC, Curtis ML, Kobari Y, Stemmerman MB, Pritchard KA. Native LDL increases EC adhesiveness by inducing ICAM-1. Arterioscler Thromb Vasc Biol. 1996;16:585–590.[Abstract/Free Full Text]
    
16. Jongkind JF, Verkerk K, Hoogerbrugge A. Monocytes from patients with combined hypercholesterolemia-hypertriglyceridemia show increased adhesion to EC in vitro. Metabolism. 1995;44:374–378.[Medline] [Order article via Infotrieve]
    
17. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of NFb and HIV-1. EMBO J. 1991;10:2247–2258.[Medline] [Order article via Infotrieve]
    
18. Staal FJT, Roederer M, Herzenberg LA. Intracellular thiols regulate activation of NFb and transcription of HIV. Proc Natl Acad Sci U S A. 1990;87:9943–9947.
    
19. Garner MM, Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions. Nucleic Acid Res. 1981;9:3047–3060.[Abstract/Free Full Text]
    
20. Fried M, Crothers DM. Equilibria and kinetics of the lac operon interactions by PAGE. Nucleic Acid Res. 1981;9:6505–6525.[Abstract/Free Full Text]
    
21. Jialal I, Grundy SM. Effect of dietary supplementation with AT on oxidative modification of LDL. J Lipid Res. 1992;33:899–906.[Abstract]
    
22. Reaven PD, Witztum JL. Comparison of supplementation with RRR-AT and all-rac-AT in humans. Arterioscler Thromb. 1993;13:601–608.[Abstract/Free Full Text]
    
23. Adams DH, Shaw S. Leucocyte-endothelial interactions and regulation of leukocyte migration. Lancet. 1994;343:831–836.[Medline] [Order article via Infotrieve]
    
24. Duplaa C, Couffinhal T, Labat L, Moreau C, Petit-Jean M, Doutre M, Lamaziere JD, 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]
    
25. Li H, Cybulsky MI, Gimbrone MA, Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler Thromb. 1993;13:197–204.[Abstract/Free Full Text]
    
26. O'Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE. Neurovascular expression of E-selectin, ICAM-1, and VCAM-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996;93:672–682.[Abstract/Free Full Text]
    
27. Weber C, Erl W, Weber PC. Enhancement of monocyte adhesion to endothelial cells by oxidatively modified LDL is mediated by activation of CD11b. Biochem Biophys Res Commun. 1995;206:621–628.[Medline] [Order article via Infotrieve]
    
28. Ragab MS, Selvaraj P, Sgoutas DS. Oxidized Lp(a) induces cell adhesion molecule Mac-1 and enhances adhesion of the monocytic cell line U937 to cultured endothelial cells. Atherosclerosis. 1996;123:103–113.[Medline] [Order article via Infotrieve]
    
29. Cominacini L, Garbin U, Pasini AF, Davoli A, Campagnola M, Contessi GB, Pastorino AM, Cascio V. Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells. Free Radic Biol Med. 1997;22:117–127.[Medline] [Order article via Infotrieve]
    
30. Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, Medford RM. VCAM-1 gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993;92:1866–1874.
    
31. Khan BV, Parthasarathy SS, Alexander RW, Medford RM. Modified LDL and its constituents augment cytokine-activated VCAM-1 gene expression in human vascular endothelial cells. J Clin Invest. 1995;95:1262–1270.
    
32. Baeuerle PA. The inducible transcription factor NFB: regulation by distinct protein subunits. Biochim Biophys Acta. 1991;1072:63–80.[Medline] [Order article via Infotrieve]
    
33. Baeuerle PA, Baltimore D. Activation of DNA binding activity in apparently cytoplasmic precursors of NFb. Cell. 1988;53:211–217.[Medline] [Order article via Infotrieve]
    
34. Baeuerle PA, Baltimore D. A specific inhibitor of NFb transcription factor. Science. 1988;242:540–546.[Abstract/Free Full Text]
    
35. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeler D. Activated transcription factor NFb is present in the atherosclerotic lesion. J Clin Invest. 1996;97:1715–1722.[Medline] [Order article via Infotrieve]
    
36. Collins T. Endothelial NFb, and the initiation of the atherosclerotic lesion. Lab Invest. 1993;68:499–506.[Medline] [Order article via Infotrieve]
    
37. Mihm S, Ennen J, Pessara U, Kurth R, Droge W. Inhibition of HIV-1 replication and NFb activity by cysteine and its derivatives. AIDS. 1991;5:497–503.[Medline] [Order article via Infotrieve]
    
38. Suzuki YJ, Packer L. Inhibition of NFb DNA binding activity by AT succinate. Biochem Mol Biol Int. 1993;31:693–700.[Medline] [Order article via Infotrieve]
    
39. Suzuki YJ, Packer L. Inhibition of NFb DNA binding activity by AT derivatives. Biochem Biophys Res Commun. 1993;193:277–283.[Medline] [Order article via Infotrieve]
    
40. Hennig B, Toborek M, Barve SJ, Barger SW, Barve S, Mattson MP, McClain CJ. Linoleic acid activates NFb and induces NFb-dependent transcription in cultured endothelial cells. Am J Clin Nutr. 1996;63:322–328.[Abstract/Free Full Text]
    
41. Kaul N, Forman HJ. Activation of NFb by the respiratory burst of macrophages. Free Radic Biol Med. 1996;21:401–405.[Medline] [Order article via Infotrieve]
    
42. Sokoloski JA, Sartorelli AC, Rosen CA, Narayanan R. Antisense oligonucleotides to the p65 subunit of NFkb block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes. Blood. 1993;82:625–632.[Abstract/Free Full Text]
    
43. Hackman A, Abe Y, Insull W, Pownall H, Smith L, Dunn K, Gotto AM, Ballantyne CM. Levels of soluble adhesion molecules in patients with dyslipidemia. Circulation. 1996;93:1334–1338.[Abstract/Free Full Text]
    

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