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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:429-436.)
© 1997 American Heart Association, Inc.

Articles

Vitamin E Inhibits Low-Density Lipoprotein–Induced Adhesion of Monocytes to Human Aortic Endothelial Cells In Vitro

Antonio Martin; Thomas Foxall; Jeffrey B. Blumberg; ; Mohsen Meydani

From the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University (A.M., J.B.B., M.M.), Boston, Mass, and the Department of Animal and Nutritional Sciences (T.F.), University of New Hampshire, Durham.

Correspondence to Mohsen Meydani, DVM, PhD, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington St, Boston, MA 02111.

	Abstract

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Abstract Monocyte adhesion to human aortic endothelial cells (ECs) is one of the early events in the development of atherogenesis. ECs were used to investigate the role of vitamin E in human monocyte adhesion to ECs in vitro. ECs incubated with 40 to 193 mg/dL of low-density lipoprotein cholesterol (LDL) for 22 hours exhibited increasing dose-dependent adherence for untreated, isolated human monocytes (P<.05). ECs exposed to the highest dose of LDL (193 mg/dL) but pretreated with 19 µmol/L -tocopherol for 24 hours showed a trend to lower adherence for monocytes compared with nontreated ECs (4.4±1.2% versus 7.6±1.9%; P=.09). This effect of vitamin E became more significant (P<.05) when ECs were exposed to a lower level of LDL (40 mg/dL) or were pretreated with a higher level of -tocopherol (42 µmol/L) and then exposed to 80 mg/dL LDL. Presupplementation of ECs with 15, 19, and 37 µmol/L -tocopherol significantly (P<.05) reduced monocyte adhesion by 6±1%, 37±6%, and 69±17%, respectively. Levels of soluble intercellular adhesion molecule-1 (sICAM-1), one of the adhesion molecules for monocytes, increased after incubation of ECs with LDL 80 mg/dL (4.7±0.7 versus 6.4±1.2 ng/mL, respectively; P<.05). Treatment of ECs with -tocopherol (42 µmol/L) significantly reduced induction of sICAM-1 by LDL to 2.2±2.3 ng/mL. After exposure to LDL, prostaglandin I₂ production by ECs was diminished, whereas presupplementation of ECs with -tocopherol partially reversed the LDL effect. Production of interleukin-1ß was not detectable when ECs were treated with -tocopherol, LDL, or -tocopherol followed by LDL. Our findings indicate that vitamin E has an inhibitory effect on LDL-induced production of adhesion molecules and adhesion of monocytes to ECs via its antioxidant function and/or its direct regulatory effect on sICAM-1 expression.

Key Words: vitamin E • monocytes • lipoprotein • ICAM-1 • prostaglandin I₂ • interleukin-1

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Significant advances have been made in the past decade in our understanding of lipoprotein metabolism and the biology of cells in the arterial wall. However, the biochemical mechanisms involved in the initiation and evolution of atherosclerosis remain unclear. Although the accumulation of cholesterol in foam cells is one of the earliest events in atheroma formation, the mechanisms by which increased levels of LDL lead to an accumulation of cholesterol in the intima are undefined. Atherosclerosis impairs EC functions, including the production of endothelium-derived relaxing factor and expression of cytokines, adhesion molecules, and growth factors.^{1 2} These changes in ECs attract and activate the transendothelial migration of monocytes.^{3 4} Studies in nonhuman primates⁵ and other animal models^{6 7 8} have demonstrated that monocyte attachment to ECs, migration, and subendothelial localization are early events in the pathogenesis of atherosclerosis. In response to some chemotactic stimuli, monocytes adhere to arterial ECs and migrate into the intima, where they take up lipids and become foam cells.⁹

Hypercholesterolemia is one of the most important risk factors for atherosclerosis and related occlusive vascular disease.¹⁰ Hypercholesterolemia has been reported to produce EC alterations with subsequent changes in membrane function and increased permeability to LDL.¹⁰ ECs stimulated by IL-1ß express adhesion molecules including sICAM-1, a transmembrane protein that mediates the adhesion of leukocytes. Monocytes adhere to ECs chiefly via the lymphocyte function–associated antigen-1 receptor.^{11 12} Interestingly, monocytes from hypercholesterolemic patients adhere more avidly to ECs than do those from healthy subjects.¹³ Treatment with the antioxidant drug probucol has been shown to reduce the adhesion of monocytes to arteries in hypercholesterolemic rabbits.¹⁴

Epidemiological and clinical studies indicate that dietary vitamin E significantly reduces the risk of cardiovascular disease.^{15 16 17 18} Reports indicate that dietary vitamin E supplementation increases cellular and tissue levels of vitamin E.^{19 20 21} Supplementing diets of rabbits with vitamin E has been shown to increase their arterial vitamin E content by several fold.²² In vitro addition of -tocopherol to the culture medium increased -tocopherol concentration in ECs.^{23 24} Vitamin E serves as the principal antioxidant in the lipid bilayer of cell membranes, protecting them against oxidative damage. In addition, vitamin E can form stable physicochemical complexes within the lipid bilayer and can modulate eicosanoid and cytokine production and signal-transduction pathways.^{25 26} These and other mechanisms may play an important role in monocyte-EC interactions. This study was designed to investigate the effect of vitamin E on EC expression of adhesion molecules and monocyte adhesion to ECs under basal conditions and in response to exposure to LDL.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
EC Culture
Human aortic ECs were purchased from Clonetics Laboratories and cultured in M-199 medium (Gibco) containing 5% FBS (Sigma Chemical Co). The culture media also contained 5 µg/mL EC growth factor prepared from bovine retina, 100 µg/mL heparin (Sigma), 100 U/mL penicillin, 100 U/mL streptomycin, and 1.25 µg/mL amphotericin B (Sigma). Cells were seeded into T-25 flasks (Corning) coated with 2% gelatin; grown to confluence in 5% CO₂, 20% O₂, and balance N₂ at 37°C; and passaged according to standard procedures with the use of 0.05% trypsin-EDTA (Sigma). The culture medium was replaced every 2 days until the cells attained confluence. ECs were seeded into gelatin-coated six-well plates (Corning), examined by phase-contrast microscopy, and photographed to monitor cell growth and confluence. The 24-hour postconfluent ECs at passage 6 were used for all experiments. ECs were characterized by the presence of von Willebrand factor antigen by use of immunofluorescence microscopy. The ECs used in this study were in perfect condition as determined by microscopic examination and cell viability (96% to 100%) at early passage as well as at passages 4 to 7. These cells were originated from the aorta of middle-aged, healthy accident victims. The isolated ECs had been tested by the vendor (Clonetics) for sterility, mycoplasma, HIV and hepatitis B virus infection, normal morphology, and rate of proliferation. After treatment of ECs with 0.05% trypsin-EDTA for 3 minutes or until 80% of the cells were detached, cell viability was determined by use of a trypan-blue exclusion test.

Vitamin E Measurement
Vitamin E (-tocopherol) content of ECs was measured by reverse-phase HPLC. Briefly, after sonication, ECs were saponified with 30% KOH in the presence of 2% pyrogallol (Sigma) at 40°C for 30 minutes. Tocol (a gift from Hoffmann-La Roche, Neutley, NJ) was added to the mixture as an internal standard. Tocopherols were extracted into 2.5 mL of hexane containing 0.002% BHT, dried under a stream of nitrogen gas, and reconstituted in 40 µL of methanol. Tocopherols were separated by HPLC by use of a 3-µm C18 reverse-phase column (Perkin-Elmer) with 100% methanol as the mobile phase. Eluted peaks were detected with a Perkin-Elmer 650-15 fluorescence spectrophotometer set at 292-nm excitation and 330-nm emission.²⁷ Peaks were integrated with a Waters 860 system. For the determination of -tocopherol concentration in the media, 100 µL of culture medium was mixed with 100 µL of ethanol containing tocol as an internal standard and extracted with 0.5 mL of hexane containing 0.002% BHT, followed by HPLC analysis.

Vitamin E Supplementation of Human Aortic ECs
A stock solution of 10 mmol/L d--tocopherol (Kodak Chemical) was made in absolute ethanol and stored at -70°C. ECs were supplemented with d--tocopherol by drying the required amount of d--tocopherol stock solution and redissolving it in 100% ethanol to achieve a final concentration of 0.05% ethanol in the culture media. The d--tocopherol–ethanol solution was mixed with FBS (10% final concentration in the media) and incubated at 37°C for 15 minutes while mixing gently every 5 minutes. The FBS containing d--tocopherol and ethanol was mixed with M-199 culture media, and ECs were incubated in this medium for 24 hours.

Isolation of LDL
LDL (1.019 to 1.063 g/mL) was isolated from plasma by single vertical, discontinuous, density gradient ultracentrifugation as described by Chung et al.²⁸ Heparinized blood was obtained from normolipidemic healthy men (LDL <160 mg/dL) and centrifuged for 15 minutes at 2500 rpm. The density of the plasma was adjusted to 1.21 g/mL by the addition of KBr (0.365 g/mL). Tubes loaded with sample and gradient were immediately placed in a near-vertical tube–90 rotor (NVT-90, Beckman Instruments) and centrifuged in a Beckman L7-80M ultracentrifuge at 70 000 rpm at 7°C for 90 minutes with slow acceleration and deceleration modes. This procedure yields three lipoprotein fractions with VLDL at the top, LDL in the upper-middle, and HDL in the lower-middle portion of the tube.²⁸ The isolated LDL was dialyzed against 150 mmol/L NaCl, 1 mmol/L EDTA (pH 7.4), filtered through a 0.2-µm filter, and stored at 4°C under nitrogen until used within 4 hours after isolation. Protein was measured by the method of Lowry et al,²⁹ with bovine serum albumin used as a standard. The concentration of cholesterol in isolated LDL was measured by standard laboratory methods with the use of a commercial diagnostic kit (Boehringer Mannheim Corp). During the isolation and purification of LDL, special precautions were taken to avoid LDL oxidation. No PC-OOH could be detected in isolated LDL (see below).

Measurement of PC-OOH
Aliquots of LDL were extracted with redistilled chloroform:methanol (2:1, vol/vol) containing 0.02% BHT. After centrifugation, the bottom layer was collected and dried in a rotary evaporator. The residue was then reconstituted in 60 µL of chloroform:methanol (2:1, vol/vol), and 20 µL was injected into the HPLC system. Chemiluminescence was measured with a Tohoku CL-110 chemiluminescence detector (Tohoku Electronic Ind).³⁰ The PC-OOH standard was synthesized from L--phosphatidylcholine,ß-linoleoyl--palmitoyl (Sigma) with visible condensed light and rose bengal used as a photosensitizer to generate singlet oxygen.³¹ The concentration of the standard was calibrated with cumene hydroperoxide with the use of potassium iodide and the starch reaction.³¹ A calibration curve was generated by injecting different concentrations of PC-OOH standard into the HPLC-chemiluminescence system with each set of LDL analyses for PC-OOH. The sensitivity of this measurement is <40 pmol/mL.

Isolation of Human Monocytes
Human peripheral monocytes were isolated as follows: 5 vol of heparinized fresh blood from fasting normolipidemic subjects was mixed with 1 vol of 6% dextran T-500 (Pharmacia Biotech AB) in 0.15 mol/L NaCl.³² After incubation at room temperature for 30 minutes, 6 vol of the upper phase (leukocyte-rich plasma) was layered onto 3 vol of Fico/Lite (Atlanta Biologicals) solution with a density of 1.068 g/mL. After centrifugation at 600g for 15 minutes at 20°C, the monocyte band was collected by aspiration. Cells isolated by this method consisted of >90% monocytes with a yield of >60%.^{32 33} Purity of the monocytes was determined by flow cytometry using antibody against CD-14 (Becton Dickinson).

Experimental Design
Confluent ECs grown in 6-well culture plates (to study the effect of LDL on monocyte adhesion and PGI₂ and IL-1ß production) or 24-well culture plates (for monocyte adhesion) were incubated in M-199 medium containing 0 to 42 µmol/L -tocopherol (average human plasma range from 12 to 30 µmol/L) for 24 hours. After EC enrichment with -tocopherol, medium was removed, cells were washed twice with M-199, and new medium containing different concentrations of LDL (56 to 193 mg/dL LDL cholesterol) was added and the cells were incubated for 22 hours. Cell supernatant was collected, and aliquots for PGI₂, IL-1ß, and sICAM-1 measurement were stored at -70°C. Subsequently, cells were washed twice with HBSS and incubated with new media containing 10⁶ human monocytes per milliliter labeled with ¹¹¹In. Monocyte adhesion was determined as described below. In experiments that used 6-well plates, three replicates were used per treatment, and in experiments that used 24- or 96-well plates, six replicates were used for each experiment. Each experiment was repeated two to three times as denoted in the footnotes of the figures and the Table.

View this table: [in this window] [in a new window]   Table 1. Effect of LDL Cholesterol and -Tocopherol on PG I2 Production by Human Aortic ECs

Monocyte Adhesion
Isolated monocytes were labeled with ¹¹¹In (Medi-Physics) by incubating cells in HBSS containing 15 to 20 µCi of ¹¹¹In (1 mCi/mL) per 10 mL of original blood volume using an orbital shaker bath at 37°C for 15 minutes. The mixture was then centrifuged at 150g for 10 minutes, and the supernatant with unincorporated label was removed. Monocytes were resuspended in the original volume of whole blood using M-199, added to the confluent human aortic EC monolayers, and incubated under rotary conditions at 37°C for 1 hour. After incubation, the supernatant was collected, monolayers were washed twice with HBSS, and the wash was combined with the supernatant. The ECs were solubilized with 1 mL of 10 mmol/L NaOH-0.1% SDS and collected. Wells were washed twice with 1 mL of HBSS, and solubilized cells and wash were combined. The amount of ¹¹¹In was determined in a Beckman 550 gamma counter (Beckman Instruments). The percentage of monocytes adhering to the ECs was calculated by dividing the total number of counts added by the number of counts associated with cells (x100).³³

Measurement of sICAM-1
sICAM-1 released into the culture media was analyzed by an ELISA (Bio-Teck Instruments, Inc) using a kit (R&D Systems). The assay involves the simultaneous reaction of sICAM-1 present in the sample or standard to two antibodies directed against different epitopes on the sICAM-1 molecule. One antibody is coated onto the walls of the microtiter well and the other is conjugated to the enzyme horseradish peroxidase. After addition of diluted samples or standards, the plate was covered and incubated for 1.5 hours at room temperature. The supernatant was removed, wells were immediately washed, tetramethylbenzidine was added, and the wells were incubated for 30 minutes at room temperature. The reaction was stopped by adding 100 µL of 4N sulfuric acid, and the optical density of each well was determined within 30 minutes using a microtiter plate reader set at 450 nm with a correction wavelength of 620 nm. The concentration of sample was determined by calculating the concentration of sICAM-1 corresponding to the mean absorbance from the standard curve and corrected for the dilution factor. Each treatment was performed in triplicate.

PGI₂ and IL-1ß Determination
PGI₂ was analyzed by radioimmunoassay for 6-keto-PGF_1, the main hydration product of PGI₂, as described by Hwang et al.³⁴ IL-1ß in the medium was measured by radioimmunoassay according to Endres et al.³⁵

Statistical Analysis
The Student's t test was used to assess the significance of the differences of the measured parameters between treated and untreated ECs.

	Results

Top Abstract Introduction Methods Results Discussion References

 
-Tocopherol Supplementation of ECs
Incubation of ECs with medium containing 0 to 58 µmol/L -tocopherol for 24 hours increased cellular -tocopherol concentration in a dose-dependent fashion, as we have observed previously.³⁶

Effect of LDL and -Tocopherol on Monocyte Adhesion
After incubation of ECs for 22 hours with 56 to 193 mg/dL LDL cholesterol, adhesion of monocytes to ECs was significantly increased (Fig 1). The adhesion of monocytes to ECs without preexposure to LDL (control) was 2.4±0.2%. After exposure of ECs to 56, 96, and 193 mg/dL LDL cholesterol, adhesion of monocytes increased to 3.8±0.5%, 4.9±1.2%, and 7.6±1.9%, respectively (Fig 1). LDL did not contain any existing lipid hydroperoxides during the incubation with ECs, as determined by a sensitive assay for phosphatidylcholine hydroperoxides.³⁰ Preincubation of ECs with 7 and 19 µmol/L -tocopherol reduced adhesion of monocytes to ECs by 18±6% and 42±8%, respectively, when cells were exposed to LDL at concentrations of 193 mg/dL LDL cholesterol (Fig 2). However, this reduction was not statistically significant (4.4±1.2% versus 7.6±1.9%, P=.09). This experiment was repeated with lower concentrations of LDL and higher concentrations of -tocopherol. Adhesion of monocytes to ECs preexposed to 40 and 80 mg/dL LDL cholesterol increased from 4.4±0.6% in the control (no added LDL cholesterol) to 4.9±0.7% and 6.2±1.2% (P<.05), respectively (Fig 3A). When ECs were enriched with -tocopherol by preincubation with medium containing 21 or 42 µmol/L -tocopherol, monocyte adhesion decreased significantly (P<.005) by 36% and 84% (from 4.4±0.6% to 2.8±1% and 0.7±0.2%) (Fig 3B). This effect of -tocopherol alone on monocyte adhesion was further tested in another experiment. Preincubation of ECs with increasing concentrations of -tocopherol in the medium at 15, 19, and 37 µmol/L significantly (P<.05) inhibited monocyte adhesion to ECs in a dose-dependent fashion (Fig 3C). When ECs were preincubated with 42 µmol/L -tocopherol, then exposed to 80 mg/dL LDL cholesterol, adhesion of monocytes was significantly lower than for nonsupplemented ECs (1.5±0.5% versus 6.2±1.2%; P<.05) (Fig 3D).

View larger version (16K): [in this window] [in a new window]   Figure 1. The effect of preexposure of human aortic ECs to increasing concentrations of LDL cholesterol on monocyte adhesion. Confluent ECs were incubated with different concentrations of LDL cholesterol in M-199 medium for 22 hours, then washed and incubated for 1 hour with human monocytes labeled with 111In. The percent of monocytes adhered to ECs was determined as described in "Methods." Bars represent mean±SD of three experiments with three replicates per treatment in each experiment. *Significantly different from control (P<.05).

View larger version (19K): [in this window] [in a new window]   Figure 2. The effect of preenrichment of human aortic ECs with -tocopherol (T) on EC adherence to monocytes after LDL exposure. Confluent ECs with or without -tocopherol presupplementation were exposed to 193 mg/dL LDL for 22 hours. Bars represent mean±SD of three experiments with three replicates per treatment in each experiment. *Significantly different from control (P<.05).

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of LDL cholesterol and -tocopherol on human aortic EC adherence to monocytes. Confluent ECs were exposed to LDL cholesterol: A, 40 and 80 mg/dL LDL for 22 hours or incubated with different concentrations of -tocopherol (A); B and C, incubated with different concentrations of -tocopherol for 24 hours; D, presupplemented with -tocopherol (21 and 42 µmol/L), then exposed to 80 mg/dL LDL for 22 hours. After these treatments, cells were washed and incubated with human monocytes labeled with 111In for 1 hour. Adherence of monocytes to ECs was determined as described in "Methods." Bars represent mean±SD of five experiments with four replicates per treatment in each experiment. -T indicates -tocopherol. *Significantly different from control (P<.05).

Effect of LDL and -Tocopherol on sICAM Levels
ECs exposed to 80 or 193 mg/dL LDL cholesterol released significantly (P<.05) more sICAM-1 (6.4±1.2 and 10.3±1.2 ng/mL, respectively) than control ECs (4.7±0.8 ng/mL) (Fig 4). Preincubation of ECs with 42 µmol/L -tocopherol reduced sICAM-1 production when cells were exposed to 80 mg/dL LDL cholesterol (2.2±2.3 versus 6.4±1.2 ng/mL; P<.05) (Fig 4). Production of sICAM-1 by ECs under basal conditions without added LDL cholesterol was reduced by 90% by presupplementation of ECs with 42 µmol/L -tocopherol (4.7±0.8 versus 0.8±0.3 ng/mL; P<.002) (Fig 5).

View larger version (46K): [in this window] [in a new window]   Figure 4. Effect of LDL cholesterol and -tocopherol (T) on human aortic EC production of sICAM-1. Confluent ECs with and without presupplementation with -tocopherol (42 µmol/L) were incubated with 80 and 193 mg/dL LDL in M-199 for 22 hours. sICAM-1 in the supernatant was measured by ELISA as described in "Methods." Bars represent mean±SD of two experiments with six replicates per treatment in each experiment. *Significantly different from control (P<.05).

View larger version (29K): [in this window] [in a new window]   Figure 5. The effect of -tocopherol (T) supplementation on human aortic EC production of sICAM-1. Confluent ECs were presupplemented with 42 µmol/L -tocopherol in M-199 for 24 hours. Production of sICAM-1 in the supernatant was measured by ELISA as described in "Methods." Bars represent mean±SD of two experiments with six replicates per treatment in each experiment. *Significantly different from control (P<.05).

Effect of LDL and -Tocopherol on PGI₂ Production by ECs
ECs incubated with LDL showed a tendency to produce less PGI₂ than control (Table). In contrast, ECs incubated with -tocopherol showed an increase in PGI₂ production. For example, incubation of ECs with 42 µmol/L -tocopherol increased PGI₂ production relative to control (114±63 versus 79±49 pg/mL; Table), although this increase was not statistically significant. In contrast, treatment of ECs with LDL reduced PGI₂ production, an effect partially reversed by -tocopherol pretreatment (Table).

Effect of LDL and -Tocopherol on IL-1ß Production by ECs
IL-1ß was not detected when ECs were incubated with M-199 media and 5% FBS or supplemented with -tocopherol or after incubation with LDL cholesterol.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we found that in vitro enrichment of human aortic ECs with -tocopherol significantly reduced EC adherence to monocytes. After supplementation of ECs with 21 to 42 µmol/L -tocopherol, adhesion of monocytes was reduced in a dose-dependent fashion. In addition, we observed that exposure of ECs to increasing concentrations of LDL cholesterol increased the adhesion of monocytes in a dose-dependent manner. The adhesion of monocytes to ECs after EC exposure to LDL cholesterol was significantly decreased by supplementing the ECs with -tocopherol.

These changes in EC adherence to monocytes by LDL cholesterol and -tocopherol treatments were accompanied by changes in sICAM-1 release from ECs. Exposure to LDL cholesterol increased sICAM-1 production by ECs, whereas supplementation of ECs with -tocopherol inhibited the production of sICAM-1. Because sICAM-1 is one of the chemotactic proteins that mediate EC and monocyte interaction, the decrease in EC adherence observed with -tocopherol enrichment alone or followed by LDL cholesterol exposure may be attributed to a decreased expression of adhesion molecules such as sICAM-1, as we found in these experiments.

The effect of LDL cholesterol on EC adherence to monocytes may be a crucial event and biologically relevant to the initiation of atherosclerosis. However, the mechanisms by which elevated concentrations of LDL cholesterol increase EC adherence to monocytes are yet to be fully determined. ECs in the presence of elevated concentrations of LDL may release nitric oxide, nitric oxide–derived metabolites, superoxide, and thiols.³⁷ Thiols in the presence of metal ions are auto-oxidized to form thyl radicals and superoxide, which can initiate lipid peroxidation and oxidative modification of LDL.³⁷ Minimally modified or oxidized LDL can stimulate the signal-transduction pathways that are involved in the expression of adhesion molecules and induce the release of chemotactic proteins from the vessel wall, resulting in monocyte adhesion and migration from the blood into the arterial intimal space.^{38 39 40} However, changes in the monocytes themselves induced by hypercholesterolemia can also be a significant contributing factor to monocyte recruitment to the arterial wall.⁴¹ For example, monocytes from hypercholesterolemic patients are larger, display a marked ruffling of the plasma membrane, and show greater adhesiveness to ECs.⁶ Furthermore, incubation of monocytes with LDL has been shown to increase adhesion to HUVECs.⁴² In the present study, we have focused on the changes in EC adherence to human monocytes effected by vitamin E and LDL cholesterol.

Interestingly, although incubation of LDL with ECs for 22 hours increased adherence of cells to monocytes, it did not oxidize LDL as measured with a sensitive and specific method for PC-OOH. Thus, it appears that peroxidation of fatty acids of phosphatidylcholine in our in vitro cell-culture system does not occur in LDL particles and therefore does not contribute to the increased adherence of ECs to monocytes. It has been reported that incubation of ECs with high concentrations of native LDL for 6 hours increases monocyte binding.⁴³ These results are supported by studies showing that an atherogenic diet in rabbits rapidly induces VCAM-1 before foam-cell formation.⁴⁴ Furthermore, when ECs were exposed to oxidized or minimally modified LDL, only minimally modified LDL increased monocyte adhesion.^{45 46} Other in vitro studies could not detect EC production of classic adhesion molecules by ECs when exposed to oxidized LDL.^{38 46} However, Kume et al⁴⁷ reported that lysophosphatidylcholine, a component of oxidized LDL, induces VCAM-1 and ICAM-1 in cultured rabbit arterial ECs. In our study, we also observed increased production of sICAM-1 by ECs after incubation with nonoxidized LDL. It is plausible that a very small amount of lysophosphatidylcholine is formed in LDL during incubation that was undetectable with presently available analytical techniques.

Our recent observations also indicate that using M-199 medium in an EC culture does not support either cholesterol ester hydroperoxides or conjugated diene production, nor did we observe any changes in electrophoretic mobility of LDL after incubation of LDL in our cell culture. However, formation of other modified molecules in LDL during incubation or a direct effect of LDL cholesterol on EC redox status cannot be ruled out. Several oxidation products of cholesterol have been indicated to play important roles in the development of atherosclerosis.⁴⁸ Recently, it has been demonstrated that dietary-induced hypercholesterolemia in rabbits enhanced the vascular release of superoxide radicals and impaired the action of endothelium-derived relaxing factor.^{4 49} It is plausible that the presence of a very low level of cholesterol oxides in LDL of a dietary origin or formed in vivo or during incubation with ECs may have contributed to our present observation. Thus, in addition to changes occurring in the LDL particle, elevated LDL cholesterol above physiological concentrations (>130 mg/dL) may be sufficient to induce activation of ECs, leading to the expression of chemotactic molecules involved in the adhesion of monocytes, and to impair EC function. However, under in vivo conditions, several components of plasma, including other lipoproteins, fat- and water-soluble antioxidants, and other cells participating in the pathogenesis of atherosclerosis, may have a modulatory effect in the adhesion of monocytes to ECs and the progression of lesions in addition to LDL.

Nevertheless, our data demonstrating a reduction of LDL-induced monocyte adhesion by -tocopherol and a decrease of sICAM-1 production by ECs support the important role of dietary vitamin E in the prevention of early events in the atherosclerotic process. Our in vitro observations provide further support to the epidemiological data on the role of dietary vitamin E in the prevention of cardiovascular disease.^{15 16 17 18} It is worth mentioning that IL-1–induced expression of VCAM-1 by HUVECs has been reported to be reduced by antioxidants such as pyrrolidinedithiocarbamate and N-acetyl cysteine.⁵⁰ -Tocopherol, as well as other antioxidants such as probucol and N-acetyl cysteine, has also been reported to inhibit adhesion of U937 monocytic cells to HUVECs when stimulated with agonists such as IL-1, lipopolysaccharide, thrombin, or PMA.⁵¹ Faruqi et al⁵¹ reported that -tocopherol inhibition of IL-1–induced monocytic adhesion was also correlated with reduced expression of E-selectin by HUVECs.

The endothelium plays a pivotal role in atherogenesis not only through production of adhesion molecules and chemotactic factors but also of cytokines and prostanoids. PGI₂ produced by ECs has antiaggregatory and vasodilatory effects on the vascular system and functions as a local antiplatelet agent.⁵² PGI₂ also decreases polymorphonuclear cell adhesion to ECs in vitro.⁵³ Under physiological conditions, the secretion of PGI₂ by ECs is very low,⁵⁴ but some studies have reported an increase in PGI₂ production in atherosclerosis,⁵⁵ in platelet activation,⁵⁶ and under oxidative and mechanical stress conditions,^{57 58} and one study reported a decrease of PGI₂ production by oxidized LDL.⁵⁹ Our data indicate that LDL cholesterol inhibits the EC secretion of PGI₂ (Table) without LDL cholesterol showing a preexisting or newly formed detectable PC-OOH during incubation with ECs. This is in accordance with other reports showing low vascular PGI₂ production at the site of plaque formation in diet-induced hypercholesterolemia in rabbits.⁶⁰ In our experiments, ECs supplemented with -tocopherol showed a tendency to produce more PGI₂ than nonsupplemented ECs, providing an indication for the antiatherosclerotic effect of vitamin E. Therefore, the tendency of LDL to decrease PGI₂ production by ECs may further contribute to increasing EC adherence to monocytes.⁵³

IL-1ß is a multifunctional immune/inflammatory mediator that, in addition to modulating the expression of EC adhesion molecules, is responsible for alterations leading to EC activation.⁶¹ In leukocytes, IL-1ß regulates the expression of specific proteins involved in the adhesion and subsequent migration of leukocytes into tissues.⁶² Through autocrine signaling, IL-1ß may induce EC secretion of chemotactic factors and expression of cell-surface adhesion molecules involved in the adhesion of circulating monocytes to the arterial endothelial lining.^{4 63} However, production of IL-1ß in our experiments was not affected by exposing ECs to LDL. Thus, it appears that IL-1ß autocrine signaling is not involved in the production of adhesion molecules by ECs effected by LDL exposure or -tocopherol.

In summary, -tocopherol supplementation in vitro decreases EC adherence to monocytes in part by reducing the expression of sICAM-1 and probably by modulating the secretion of PGI₂. Thus, evidence from this and other studies supports the concept that the apparent preventive effect of dietary and supplemental vitamin E in reducing cardiovascular disease can be attributed to a combined effect of this nutrient on factors that are involved in the pathogenesis of atherosclerosis, including oxidative modification of LDL, providing a cytoprotective and membrane-stabilizing action on vascular ECs, reducing the production of chemoattractant molecules, and decreasing the adherence of ECs to circulating monocytes.

	Selected Abbreviations and Acronyms

 

BHT = butylated hydroxytoluene EC = endothelial cell ELISA = enzyme-linked immunosorbent assay FBS = fetal bovine serum HBSS = Hanks' balanced salts HPLC = high-performance liquid chromatography HUVEC = human umbilical vein endothelial cell IL = interleukin 111In = 111In-oxiquinoline PC-OOH = phosphatidylcholine hydroperoxide PG = prostaglandin sICAM-1 = soluble intercellular adhesion molecule-1 VCAM = vascular cell adhesion molecule

	Acknowledgments

 
This project was supported with federal funds from the US Department of Agriculture, Agricultural Research Service under contract No. 53-3K06-0-1. The authors would like to thank Jennifer Munnis for her assistance in the preparation of the manuscript.

	Footnotes

 
The contents of this article do not necessarily reflect the views of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

Received June 19, 1995; accepted July 9, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
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