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(Circulation. 2000;102:670.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Remnant Lipoproteins Induce Proatherothrombogenic Molecules in Endothelial Cells Through a Redox-Sensitive Mechanism

Hideki Doi, MD; Kiyotaka Kugiyama, MD; Hideki Oka, MD; Seigo Sugiyama, MD; Nobuhiko Ogata, MD; Shun-ichi Koide, MD; Shin-ichi Nakamura, MD; Hirofumi Yasue, MD

From the Department of Cardiovascular Medicine, Kumamoto University School of Medicine, Kumamoto City, Japan.

Correspondence to Kiyotaka Kugiyama, MD, Department of Cardiovascular Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto City, 860-8556 Japan. E-mail kiyo{at}gpo.kumamoto-u.ac.jp

	Abstract

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Background—Triglyceride-rich lipoproteins (TGLs) are atherogenic. However, their cellular mechanisms remain largely unexplained. This study examined the effects of isolated remnant-like lipoprotein particles (RLPs) on the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and tissue factor (TF), proatherothrombogenic molecules, in cultured human endothelial cells.

Methods and Results—RLPs were isolated from plasma of hypertriglyceridemic patients by use of the immunoaffinity gel mixture of anti–apoA-1 and anti–apoB-100 monoclonal antibodies. The incubation of cells with RLPs significantly upregulated mRNA and protein expression of these molecules. Total TGLs (d<1.006) and LDL had fewer or minimal effects on expression of these molecules compared with RLPs. RLPs increased intracellular oxidant levels, as assessed with an oxidant-sensitive probe. Combined incubation with -tocopherol or N-acetylcysteine, both antioxidants, suppressed RLP-induced increase in expression of these molecules. In patients with higher plasma levels of RLPs, plasma levels of soluble forms of ICAM-1 and VCAM-1 were significantly higher than in patients with lower RLP levels. Treatment with -tocopherol for 1 month decreased levels of the soluble adhesion molecules concomitantly with an increase in resistance of RLPs to oxidative modification in patients with high RLP levels.

Conclusions—RLPs upregulated endothelial expression of ICAM-1, VCAM-1, and TF, proatherothrombogenic molecules, partly through a redox-sensitive mechanism. RLPs may have an important role in atherothrombotic complications in hypertriglyceridemic patients.

Key Words: lipoproteins • endothelium • cell adhesion molecules • antioxidants • atherosclerosis

	Introduction

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There is increasing evidence that triglycerides and triglyceride-rich lipoproteins (TGLs) carry a risk for coronary artery disease (CAD).^{1 2} However, cellular mechanisms for the relation of hypertriglyceridemia with atherosclerotic development remain largely unexplained, in contrast to hypercholesterolemia, in which oxidatively modified LDLs contribute importantly to atherogenesis.³ We recently showed that remnant-like lipoprotein particles (RLPs), isolated by an immunoseparation method, have a causative role in endothelial vasomotor dysfunction in human coronary arteries and that RLPs directly induced endothelial dysfunction in the isolated rabbit aorta.^{4 5} Thus, RLPs may have a direct role in endothelial dysfunction, an early event leading to atherosclerotic development, in hypertriglyceridemic patients. Indeed, we recently demonstrated that high RLP levels predict coronary events in patients with CAD independently of traditional coronary risk factors.⁶ Most individuals spend 12 hours daily in a postprandial state; thus, postprandial RLPs may play a more important role in atherogenesis in hypertriglyceridemic patients than fasting RLPs.

Altered or activated endothelial functions play an important role in atherogenesis through a variety of endothelium-derived proatherothrombogenic molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and tissue factor (TF).^{7 8 9} This study was thus aimed at examining the effects of isolated postprandial RLPs on endothelial production of these molecules, considering a possible relevance of RLPs in atherothrombogenesis.

	Methods

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Lipoprotein Preparations
EDTA-plasma (1 mg/mL EDTA) was obtained at 5 hours after the test meal from 18 hypertriglyceridemic subjects who had fasting RLP levels of >5.1 mg cholesterol/dL (75th percentile of the distribution of fasting RLP levels in 250 consecutive hospitalized patients in our hospital). The postprandial RLP levels were 12.2±2 mg cholesterol/dL. The energy content of the test meal was 490 kcal/m² body surface area (86.8% from fat, 8.7% from carbohydrate, and 4.5% from protein; the ratio of polyunsaturated fatty acids to saturated fatty acids was 0.51). The subjects had no serious diseases and had not taken cardiovascular medications, pharmacological doses of antioxidants, or estrogen for 7 days. Patients with familial hypertriglyceridemia were not included in the study subjects.

RLPs were routinely prepared with columns (1.5x30 cm) packed with 50 mL of immunoaffinity gel containing anti–apoA-1 and anti–apoB-100 monoclonal antibodies (Japan Immunoresearch Laboratories).^{4 5 6 10} This unique anti–apoB-100 antibody has been shown to recognize apoB-100 in LDL and most VLDLs but not in apoE-enriched VLDL.¹⁰ The plasma samples (5 mL) from each subject were applied to the columns, and the unbound fraction containing apoE-enriched lipoproteins and albumin was eluted with PBS (mmol/L: NaCl 138, KCl 2.7, NaH₂PO₄ 8.1, KH₂PO₄ 1.1; pH 7.4) at a rate of 30 mL/h for 6 hours at 4°C. The bound fraction was subsequently eluted at a rate of 30 mL/h for 3 hours with 3 mol/L NaSCN containing 1 mg/mL BSA (fatty acid–free BSA) and was immediately dialyzed against EDTA/saline (pH 7.4). The unbound and bound fractions were ultracentrifuged (d<1.006) to isolate RLPs and bound TGLs, respectively, and they were concentrated by the membrane filtration method. According to analyses^{4 5} with SDS-PAGE, elution profiles with high-performance liquid chromatography (HPLC), agarose gel electrophoretograms, electron photomicrographs, and compositions of lipids and apolipoproteins, the unbound fraction isolated by this method from plasma 5 hours after the meal consisted mainly of VLDL remnants and small amounts of chylomicron remnants (amount ratio of apoB-48 relative to apoB-100 was 0.13±0.01, determined by densitometric analysis on SDS-PAGE). Total TGLs and LDLs were isolated from the same EDTA-plasma as used for RLP isolation by ultracentrifugation (d<1.006 for total TGLs, 1.019<d<1.063 for LDLs). The prepared lipoproteins were extensively dialyzed for 24 hours at 4°C against PBS containing EDTA (50 µmol/L) and then sterilized by filtration (filter pore size, 0.22 µm; Millipore). Total cholesterol and triglycerides were measured by enzymatic methods.⁴ Final concentrations of the prepared lipoproteins were 4.5 to 6.5 mg cholesterol/mL.

In Vivo Treatment With -Tocopherol
A consecutive series of 74 patients who underwent cardiac catheterization for atypical chest pain in Kumamoto University Hospital were studied for a possible relation of RLP levels with plasma levels of soluble forms of ICAM-1 (sICAM-1) and VCAM-1 (sVCAM-1). This study excluded patients treated with lipid-lowering drugs and pharmacological doses of antioxidants 1 month before this study. Of these patients, 12 who had higher plasma levels of fasting RLPs (>5.1 mg cholesterol/dL) were randomly assigned to have oral intake of -tocopherol (300 mg/d, n=6) or placebo (n=6) for 4 weeks. They were advised to adhere to their usual diet and exercise activity throughout 4 weeks. Before and at the end of the 4 weeks of treatment, EDTA-plasma was obtained after an overnight fast (for assay of adhesion molecules) and at 5 hours after the test meal (for isolation of RLPs). The -tocopherol content of lipoproteins was measured by HPLC. Written informed consent was obtained from all patients before the study. This study was in agreement with the guidelines approved by the ethics committee at our institution.

Antigen levels of sICAM-1 and sVCAM-1 in the fasting plasma were determined before and at the end of the treatment with -tocopherol or placebo by the double antibody sandwich methods with ELISA using monoclonal antibodies against human sICAM-1 and sVCAM-1, respectively (R&D Systems).¹¹ Standard curves obtained from the assay kits showed that the lower limits of detection were 0.35 ng/mL of sICAM-1 and 2.0 ng/mL of sVCAM-1.

Cell Culture
Primary cultures of human umbilical vein endothelial cells (HUVECs) were obtained as previously described.¹² Confluent HUVECs at passage 2 were used in this study. After serum starvation for 8 hours, the medium was replaced with serum-free medium 199, and the cells were then incubated with one of the lipoprotein preparations in the presence or absence of -tocopherol or N-acetylcysteine (NAC) for the indicated times. The treated cells were assayed for measurements of expression of mRNA and protein and lipid peroxides.

Cell-Surface ELISA for Adhesion Molecules on HUVECs
After treatment in 96-well microplates, the cells were rinsed and incubated with 0.5% periodic acid at 4°C for 20 minutes for inactivation of endogenous peroxidase. Then, the cells were rinsed and treated with mouse anti-human ICAM-1 (Dako) or VCAM-1 monoclonal antibodies (Coulter-Immunotech) at 4°C for 2 hours. The cells were subsequently washed with PBS and then treated with peroxidase-conjugated goat anti-mouse IgG at 4°C for 1 hour. The plates were then washed and incubated with 0.1 mL/well of 3,3',5,5'-tetramethylbenzidine substrate (Dako) at 4°C for 30 minutes. The reaction was stopped by addition of 50 µL of 1N hydrochloric acid and 3N sulfuric acid mixture. The plates were read on an ELISA reader (M-Emax, Wako) at OD 450 nm after blanking on rows stained only with second-step antibody.¹³

Assays of Soluble Forms of Adhesion Molecules in Culture Medium
After the treatment of HUVECs in 12-well plates for 12, 24, and 36 hours, the conditioned medium was collected and then centrifuged at 15 000g for 10 minutes to remove cell debris. The levels of the soluble forms of the adhesion molecules in the culture medium were measured with the same ELISA kits as described above.

Determination of TF Protein
After treatment in 6-well plates, the cells were detached by addition of cold PBS containing 1% Triton X-100 and stirred for 12 hours at 4°C. The suspension was centrifuged to separate cell debris, and TF antigen levels in the cell extracts were measured by ELISA (Imubind TF ELISA Kit, Loxo GmbH).¹⁴

RNA Isolation and Northern Blot Analysis
Total RNA was extracted from the treated cells by the guanidine thiocyanate method.¹⁵ Northern blot analysis was then performed by loading of 20 µg of RNA in each lane of 1% agarose-formaldehyde gels, electrophoretic separation, transfer to nylon membranes (Schleicher & Schuell), and ultraviolet cross-linking. Complementary cDNA probes were ³²P-labeled by the random primer method to a specific activity of 5x10⁸ cpm/µg DNA. The cDNA probes in this study included the following: (1) a 1.8-kbp cDNA probe for ICAM-1 and a 700-bp cDNA probe for VCAM-1 (a kind gift from Dr Ron Cob, Tanabe Research Laboratories, San Diego, Calif); (2) a 641-bp cDNA probe for TF (a kind gift from Dr Lindsey A. Miles, The Scripps Research Institute, La Jolla, Calif); and (3) a 1-kb cDNA probe for GAPDH. The membranes were hybridized with the ICAM-1, VCAM-1, or TF probe. The same blot was rehybridized with the GAPDH probe to normalize the amount of ICAM-1, VCAM-1, and TF mRNA. The intensity of hybridization signals was determined by use of an FLA 2000 (Fujifilm).

Electrophoretic Mobility Shift Assay
Nuclear extraction from the treated cells and electrophoretic mobility shift assay were performed as described previously.¹⁶ The sequence of the probe for nuclear factor (NF)-B used in this study was 5'-CCAGAGGGGACTTTCCGAGAGG-3'.

Measurements of Intracellular Oxidant Levels, Lipid Peroxide Levels in the Medium, and Susceptibility of RLPs to Oxidative Modification
Intracellular levels of reactive oxygen species were measured by flow cytometric analysis using an oxidant-sensitive fluorescence probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA, Eastman Kodak) as previously described.¹⁷ After the treatment, the cells were incubated with phenol red–free medium 199 containing 5 µmol/L DCFH-DA for 30 minutes at 37°C in the dark. Then, the cells were washed 3 times with cold PBS (4°C) and detached by addition of cold PBS containing 0.05% EDTA. The suspended cells were washed and immediately analyzed with a fluorescence-activated cell sorter (FACScan, Becton Dickinson). For each analysis, 10 000 events were recorded.

Lipid peroxide levels in the culture medium were measured by colorimetric assay (Bioxytech, OXIS International, Inc).

The susceptibility of RLPs to oxidative modification was determined by measuring Cu²⁺-induced formation of conjugated dienes.¹⁸ The conjugated diene formation in RLPs (0.1 mg triglyceride/mL) was monitored by the change in absorbance at 234 nm in a spectrophotometer.

Reagents
All reagents for cell culture were from Gibco BRL. NAC, -tocopherol, and other chemicals were purchased from Sigma Chemical Co. Poly (dI-dC) was from Pharmacia Biotech Inc. [-³²P]CTP and [-³²P]ATP were from Amersham Corp. Antibody raised against p50, p65, RelB, and c-Rel were from Santa Cruz Biotechnology Inc.

Statistical Analysis
All values were expressed as mean±SEM otherwise indicated. The difference between 2 mean values was analyzed with unpaired Student’s t test. The mean values in >3 groups were compared by 1-way ANOVA, then the difference between 2 mean values was analyzed with Fisher’s protected least significant difference test. The effects of treatments on the plasma levels of soluble adhesion molecules in patients were compared by 2-way ANOVA with repeated measures followed by post hoc testing with the Scheffé test. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of RLPs on mRNA and Protein Expression of ICAM-1, VCAM-1, and TF and Levels of sICAM-1 and sVCAM-1 in Culture Medium
The incubation of HUVECs with RLPs increased mRNA levels of ICAM-1, VCAM-1, and TF, as shown in Figure 1. Furthermore, RLPs increased cell surface expression of ICAM-1 and VCAM-1 protein and TF protein levels in cell homogenates, as shown in Figure 2. The effects were dose-dependent, and similar relative values were obtained if triglycerides were used as lipoprotein concentrations (data not shown). Bound TGLs, total TGLs, and LDL had fewer or minimal effects on mRNA and protein expression of ICAM-1, VCAM-1, and TF compared with RLPs, as shown in Figures 1 and 2. Combined incubation with -tocopherol or NAC suppressed the RLP-induced mRNA and protein expression of these molecules, as shown in Figures 3 and 4. The inhibitory effects of these antioxidants were dose-dependent, ie, 50 to 150 µmol/L of -tocopherol suppressed the increase in the cell surface expression of ICAM-1 by 50% to 80%, and 0.1 to 10 mmol/L of NAC suppressed it by 50% to 70%. In human coronary artery endothelial cells (Applied Cell Biology Research Institute), RLPs also caused an increase in cell surface protein expression of adhesion molecules, which was suppressed by -tocopherol and NAC (data not shown). RLPs isolated from the patients after treatment with -tocopherol for 4 weeks had significantly less effect on mRNA and protein expression of ICAM-1 and VCAM-1 than RLPs from patients before treatment (mRNA, 43.3±6.4% and 35.0±5.4% of the respective pretreatment values; protein, 34.8±6.4% and 48.9±9.2% of the respective pretreatment values, n=6).

View larger version (43K): [in this window] [in a new window]   Figure 1. Effects of RLPs and other lipoproteins on ICAM-1, VCAM-1, and TF mRNA expression. Confluent HUVECs in a 100-mm plastic dish were treated with 1 lipoprotein preparation (0.1 mg cholesterol/mL) at 37°C for 6 hours (ICAM-1 and VCAM-1) or for 2 hours (TF). Northern blot hybridization was performed as described in Methods. Blots are representative of 8 separate experiments. Bar graph shows relative amounts of ICAM-1, VCAM-1, and TF mRNA expression to GAPDH mRNA from 8 separate experiments. Data (mean±SEM) are normalized to respective control values. *P<0.001 vs control; P<0.001 vs bound TGLs, total TGLs, and LDL.

View larger version (21K): [in this window] [in a new window]   Figure 2. Effects of RLPs and other lipoproteins on cell expression of ICAM-1, VCAM-1, and TF protein. Confluent HUVECs were treated with 1 lipoprotein preparation (0.1 mg cholesterol/mL). Values are mean±SEM from 8 separate experiments. *P<0.05, **P<0.005 vs control; P<0.001 vs bound TGLs, total TGLs, and LDL.

View larger version (48K): [in this window] [in a new window]   Figure 3. Effects of antioxidants on RLP-induced upregulation of ICAM-1, VCAM-1, and TF mRNA expression. Confluent HUVECs in a 100-mm plastic dish were treated with RLPs (0.1 mg cholesterol/mL) in presence or absence of -tocopherol (100 µmol/L, RLPs+-tocopherol) or NAC (1 mmol/L, RLPs+NAC) at 37°C for 6 hours (ICAM-1 and VCAM-1) or for 2 hours (TF). Blots and bar graph are shown as in Figure 1. *P<0.01, **P<0.005 vs control; P<0.005 vs RLPs.

View larger version (22K): [in this window] [in a new window]   Figure 4. Effects of antioxidants on RLP-induced upregulation of cell expression of ICAM-1, VCAM-1, and TF protein. Confluent HUVECs were treated with RLPs (0.1 mg cholesterol/mL) in presence or absence of -tocopherol (100 µmol/L, RLPs+-tocopherol) or NAC (1 mmol/L, RLPs+NAC). Values are mean±SEM from 8 separate experiments. *P<0.001 vs control; P<0.001 vs RLPs.

RLPs (0.2 mg cholesterol/mL) increased the levels of sICAM-1 and sVCAM-1 in the culture medium in a time-dependent manner, but the combined incubation with -tocopherol (20 µmol/L) and NAC (10 mmol/L) suppressed the RLP-induced increase in these levels (sICAM-1 and sVCAM-1: 24 hours after incubation with PBS, 1.4±0.1 and 4.8±0.1 ng/mL; with RLPs alone, 2.6±0.2* and 6.0±0.2* ng/mL; with RLPs+-tocopherol, 1.6±0.1# and 5.2±0.1# ng/mL; with RLPs+NAC, 1.5±0.1# and 5.4±0.1# ng/mL, respectively; n=6 in each experiment; *P<0.01 versus PBS, #P<0.05 versus RLPs alone).

Endotoxin levels, measured by the Limulus assay, were very low in all of the lipoprotein preparations, namely, <10 pg/0.1 mg cholesterol of lipoprotein, at which level there was no significant effect on the protein expression of the adhesion molecules.

Quantification of Lipid Peroxides in Cells and in Culture Medium
In flow cytometric analysis, 2',7'-dichlorofluorescein fluorescence intensity was increased in the cells after 1 hour of treatment with RLPs (0.1 mg cholesterol/mL) compared with the time-control cells (mean peak flow intensities were 19.2±1.9 [control] versus 65.5±2.9 [RLPs], n=6, P<0.001) (Figure 5). The increase in the fluorescence intensity was suppressed by combined incubation with -tocopherol (100 µmol/L) (mean peak flow intensities were 65.5±2.9 [RLPs] versus 39.6±1.3 [RLPs+-tocopherol], n=6, P<0.001) (Figure 5). Incubation of HUVECs with RLPs (0.1 mg cholesterol/mL) increased lipid peroxide levels in the culture medium (125.9±2.6 nmol · L^-1 · 6 h^-1 before the incubation versus 385.1±59.1 nmol · L^-1 · 6 h^-1 after the incubation, n=6, P<0.001).

View larger version (17K): [in this window] [in a new window]   Figure 5. Quantification of intracellular oxidant levels in endothelial cells. After treatment with RLPs (0.1 mg cholesterol/mL) in presence or absence of -tocopherol (100 µmol/L) for 1 hour, cells were incubated with phenol red–free medium 199 containing 5 µmol/L DCFH-DA for 30 minutes at 37°C in the dark. Intracellular levels of reactive oxygen species were analyzed as described in Methods.

Effects of RLPs on DNA-Binding Activity of NF-B
Incubation of the cells with RLPs for 90 minutes increased DNA-binding activity of NF-B, as shown in Figure 6A. Combined incubation with -tocopherol attenuated the NF-B activation by RLPs (Figure 6B).

View larger version (73K): [in this window] [in a new window]   Figure 6. Effect of RLPs on DNA-binding activity of NF-B. Confluent HUVECs in a 100-mm plastic dish were treated with RLPs (0.1 mg cholesterol/mL) in presence or absence of -tocopherol (100 µmol/L) at 37°C for 90 minutes. Nuclear extracts from treated cells were analyzed for protein binding to NF-B consensus sequence by electrophoretic mobility shift assay (5% gel). Black arrow indicates NF-B/DNA complex. A, Lane 1, cells were treated with PBS (time control); lane 2, RLPs; lane 3, RLPs plus cold NF-B oligonucleotide probe; lanes 4 through 7, supershift assays of RLP-induced NF-B/DNA complex using antibodies against p50, p65, c-Rel, and RelB. Gray arrow indicates supershifted band (lanes 4 and 5). B, Lane 1, PBS; lane 2, RLPs; lane 3, RLPs plus -tocopherol.

Effects of In Vivo Treatment With -Tocopherol on Plasma Levels of Adhesion Molecules
Plasma levels of sICAM-1 and sVCAM-1 were higher in patients with higher RLP levels (>5.1 mg cholesterol/dL, 75th percentile of the RLP distribution, n=12) than those with lower RLP levels (<2.4 mg cholesterol/dL, 25th percentile, n=12) (sICAM-1, 406±34 ng/mL in patients with higher RLP levels versus 231±19 ng/mL in patients with lower RLP levels, P<0.001; sVCAM-1, 659±39 ng/mL in patients with higher RLP levels versus 462±29 ng/mL in patients with lower RLP levels, P<0.001). Treatment with -tocopherol for 4 weeks significantly suppressed plasma levels of both adhesion molecules in patients with higher RLP levels (sICAM-1, 406±35 ng/mL before treatment versus 326±20 ng/mL after treatment, n=6, P<0.001; sVCAM-1, 648±53 ng/mL before treatment versus 559±33 ng/mL after treatment, n=6, P<0.001, by 2-way ANOVA), whereas placebo treatment had no effect (sICAM-1, 379±32 ng/mL before treatment versus 361±43 ng/mL after treatment, n=6, P=NS; sVCAM-1, 615±39 ng/mL before treatment versus 613±33 ng/mL after treatment, n=6, P=NS). The lag time of the oxidation of RLPs isolated from patients under treatment with -tocopherol was longer than that of RLPs before treatment (438±98 versus 253±21 minutes, respectively, n=6, P<0.05), indicating that administration of -tocopherol effectively exerted an antioxidant effect on the isolated RLPs. Neither treatment with -tocopherol nor placebo affected lipid profiles and hemodynamics (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study showed that RLPs upregulated endothelial expression of ICAM-1 and VCAM-1, which are responsible for monocyte recruitment into the arterial walls,^{7 8} and TF, which is essential for thrombotic events,⁹ at the same range of RLP concentrations as in peripheral plasma in patients with CAD (0.05 to 0.5 mg cholesterol/mL). Thus, high plasma levels of RLPs may have an important role in development of atherosclerosis and thrombotic events through endothelial upregulation of these proatherothrombogenic molecules. Furthermore, the present study showed that RLPs increased the endothelial release of sICAM-1 and sVCAM-1 into the culture medium and that plasma levels of both sICAM-1 and sVCAM-1 were increased in patients with higher RLP levels compared with those with the lower levels. Although the tissue sources of plasma sICAM-1 and sVCAM-1 are multiple, the plasma levels might be derived partly from the activated vascular endothelium.^{8 11 13 19} Thus, these data may support potentially stimulatory effects of RLPs on endothelial expression of adhesion molecules and their release in vivo in patients with high RLP levels. The present study further showed that RLPs increased intracellular oxidant levels in the cultured cells and that -tocopherol and NAC suppressed RLP-induced increases in mRNA and protein expression of ICAM-1, VCAM-1, and TF. These results suggest that the induction of these molecules by RLPs was at least partly mediated by redox-sensitive mechanisms.

Several mechanisms for the RLP-induced increase in oxidative stress in endothelial cells can be considered. During incubation of endothelial cells with RLPs, endothelium-derived reactive oxygen species may initiate and propagate a chain of free radical reactions, especially in polyunsaturated fatty acids in RLPs, leading to production of highly reactive intermediates that could, in turn, be transferred from RLPs to endothelial cells.^{20 21} This sequence of events may cause an increase in oxidative stress in the cultured endothelial cells. This mechanism is supported by the present results showing the increase in intracellular oxidant levels and in lipid peroxide levels in the culture medium containing RLPs after the incubation of the endothelial cells with RLPs. In patients with high levels of remnant lipoproteins, remnants can flow into the subendothelial space, where the same interaction between remnants and endothelial cells as observed in the present in vitro study could occur and result in the endothelial upregulation of these redox-sensitive molecules. Indeed, the present study further showed that administration of -tocopherol, an antioxidant, decreased plasma levels of both sICAM-1 and sVCAM-1 in patients with high RLP levels concomitantly with the increase in resistance of RLPs to Cu²⁺-induced oxidation. RLPs isolated from the patients under treatment with -tocopherol also had fewer effects on mRNA and protein expression of adhesion molecules in the cultured cells.

The present study did not examine the transcriptional activity of these genes and the downstream signals leading to the upregulation of these proatherothrombogenic genes by oxidant stress. However, these genes are known to have oxidative stress–responsive elements, such as NF-B, in their promoter/enhancer regions.^{22 23} The present study showed that RLPs activated the DNA-binding activity of NF-B in endothelial cells, which was inhibited by coincubation with -tocopherol. Thus, it is possible that RLPs may cause an increase in oxidative stress, leading to transcriptional activation of these genes in endothelial cells, possibly through a mechanism mediated by the activation of oxidative stress–responsive elements. It remains largely undetermined why the cellular effects were greater in RLPs than in total TGLs. We previously reported that RLPs induced endothelial dysfunction in an apolipoprotein receptor–independent manner.⁴ It is possible that smaller size and different lipid structure in RLPs may be partly responsible for the greater cellular effects of RLPs than of their precursors, ie, unmetabolized VLDLs and chylomicrons. Although a pathophysiological role and clinical usefulness of the RLP fraction in atherogenesis have been demonstrated,^{4 5 6 24} it is not yet established that RLPs are completely identical with remnant lipoproteins because of their heterogeneous characteristics in size and composition.

A previous in vitro experiment showed that RLPs were taken up by macrophages and caused foam cell formation.²⁴ Furthermore, RLPs caused endothelial vasomotor dysfunction, as shown in our previous reports.^{4 5} Very recently, we showed that high RLP levels predict coronary events in patients with CAD independently of traditional coronary risk factors.⁶ Thus, the previous data and the present results suggest that these proatherothrombogenic properties of RLPs may play an important role in the cardiovascular events in hypertriglyceridemic patients.

In conclusion, RLPs induced ICAM-1, VCAM-1, and TF in cultured endothelial cells through redox-sensitive mechanisms. Thus, remnant lipoproteins may have a direct and causative role in atherothrombotic development in hypertriglyceridemic patients.

	Acknowledgments

 
This study was supported in part by grant-in-aid C09670730 from the Ministry of Education, Science, and Culture and a research grant for cardiovascular diseases (7A-3, 9A-3) from the Ministry of Health and Welfare, Japan, and a Smoking Research Foundation Grant for Biomedical Research, Tokyo, Japan.

Received December 30, 1999; revision received March 1, 2000; accepted March 8, 2000.

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