Factors Influencing the Ability of HDL to Inhibit Expression of Vascular Cell Adhesion Molecule-1 in Endothelial Cells

From the University of Adelaide, Department of Medicine, Royal Adelaide Hospital (D.T.A., M.A.C., P.J.B.), the Cardiovascular Investigation Unit, Royal Adelaide Hospital (K.A.-R.), and the Hanson Centre for Cancer Research, Department of Human Immunology, IMVS (M.A.V., J.R.G.), Adelaide, South Australia, Australia.

Correspondence to Professor Philip J. Barter MBBS, PhD, Department of Medicine, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia, Australia 5000.

	Abstract

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Abstract—We have previously reported that high density lipoproteins (HDLs) inhibit the cytokine-induced expression of adhesion molecules in endothelial cells. Here we investigate whether different preparations of HDLs vary in their ability to inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) activated by tumor necrosis factor- (TNF-). HDLs collected from a number of different human subjects all inhibited VCAM-1 expression in a concentration-dependent manner, although the extent of inhibition varied widely between subjects. The inhibitory activities of the HDL₂ and HDL₃ subfractions isolated from individual subjects also differed. Whether equated for concentrations of apolipoprotein (apo) A-I or cholesterol, the inhibitory activity of HDL₃ was superior to that of HDL₂. This difference remained apparent even when the HDL subfractions were present only during preincubations with the HUVECs and were removed before activation by TNF-. To determine whether the inhibitory effect of HDL₃ was influenced by apolipoprotein composition, preparations of HDL₃ were modified by replacing all of their apo A-I with apo A-II. This change in apolipoprotein composition had no effect on the ability of the HDL₃ to inhibit endothelial VCAM-1 expression. Thus, it has been shown that different preparations of HDLs differ markedly in their abilities to inhibit VCAM-1 expression in cytokine-activated HUVECs. The mechanism underlying the differences remains to be determined.

Key Words: endothelial cells • vascular cell adhesion molecule-1 • HDL • HDL subfractions • atherosclerosis

	Introduction

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The importance of high density lipoproteins (HDLs) relates to their ability to protect against the development of coronary heart disease.¹ There are several potential mechanisms that may account for this cardioprotective function of HDLs. They may deplete atherosclerotic plaques of cholesterol by virtue of their ability to promote the efflux of cholesterol from foam cells.² They also have the capacity to reduce the atherogenicity of low density lipoproteins (LDLs) by inhibiting their oxidative modification.³ In addition, on the basis of our recent observations, HDLs may be antiatherogenic by virtue of their ability to inhibit the expression of adhesion molecules on endothelial cells.⁴

Adhesion of monocytes to the vascular endothelium is one of the earliest events in atherogenesis.⁵ This adhesion is mediated by adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, and E-selectin,⁶ all of which are rapidly synthesized by endothelial cells in response to stimulation by cytokines.⁷ The expression of endothelial cell VCAM-1 coincides with the development of early foam cell lesions in hypercholesterolemic rabbits,⁸ whereas VCAM-1, intercellular adhesion molecule-1, and E-selectin have all been detected in the arterial endothelium over existing atheromatous plaques.⁹ The concentration of soluble VCAM-1 has been reported to be elevated in the plasma of patients with atherosclerosis.¹⁰

The current study addresses the ability of HDLs to inhibit cytokine-induced expression of VCAM-1 in endothelial cells. Specifically, it seeks to determine whether HDLs from different subjects or HDLs of differing size and composition also differ in terms of their ability to inhibit adhesion molecule expression.

	Methods

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Isolation and Characterization of HDLs
Blood was collected from 6 fasting, healthy donors in tubes containing disodium EDTA (final concentration, 1 mg/mL) and placed immediately on ice. Plasma was separated by centrifugation at 4°C. HDLs were isolated by sequential ultracentrifugation at 4°C in a Beckman TLA 100.4 rotor in a Beckman TL-100 ultracentrifuge. Density adjustments were made by the addition of solid KBr. HDLs were isolated in their appropriate density range: total HDLs 1.07<d<1.21 g/mL, HDL₂ 1.07<d<1.12 g/mL, and HDL₃ 1.13<d<1.21 g/mL. Two spins were carried out at both the lower and higher densities. Each spin was at 100 000 rpm. for 17 hours. The resulting HDLs were dialyzed against 3x 1 L of endotoxin-free PBS (pH 7.4) before use in experiments. The study protocol was approved by the Royal Adelaide Hospital human ethics committee.

HDL particle size distribution was assessed by electrophoresis on 3% to 40% nondenaturing polyacrylamide gradient gels (Gradipore).¹¹ Concentrations of apolipoproteins and lipid constituents in the HDLs were determined as described previously.¹²

Isolation of HDL₂ and HDL₃ Proteins and Lipids
HDL₂ and HDL₃ were isolated as described above and delipidated as described.¹³ The protein constituents were resuspended in PBS and stored at 4°C. The lipids were extracted by the method of Folch et al¹⁴ and resuspended in PBS before being sonicated under N₂ by the method of Sparks et al¹⁵ at 51°C. The resuspended apolipoproteins and the sonicated lipid preparations were stored at 4°C and were used within 1 week.

Preparation of HDL₃ Containing Apo A-II Only (A-II HDL₃)
The apo A-I in HDL₃ was displaced with lipid-free apo A-II as described previously.¹⁶ In brief, HDL₃s were isolated by ultracentrifugation as described above. Lipid-free apo A-II was added to the HDL₃ at an apo A-II to apo A-I molar ratio of 2:1. As a control, another aliquot of HDL₃ was supplemented with PBS in place of apo A-II. Both samples were maintained at room temperature for 30 minutes and then reisolated as the supernatant after ultracentrifugation at 1.21 g/mL. The samples were dialyzed against 3x 1 L of endotoxin-free PBS before use.

Preparation of Discoidal Reconstituted HDLs
Discoidal reconstituted HDLs (rHDLs) containing apo A-I and 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC) were prepared by the cholate dialysis method described by Matz and Jonas.¹⁷

Human Umbilical Vein Endothelial Cells (HUVECs)
HUVECs were isolated as described previously.¹⁸ Cells were cultured on gelatin-coated culture flasks in medium M199 with Earle's salts (Trace Biosciences) supplemented with 20% FCS (Commonwealth Serum Laboratories), 20 mmol/L HEPES, 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, nonessential amino acids, penicillin, streptomycin, 20 mg/mL endothelial growth supplement (Collaborative Research), and 20 mg/mL heparin (Sigma Chemical Co).

Incubation Conditions
Confluent preparations of passage 3, 4, or 5 HUVECs were incubated for 1 hour in the presence of the various preparations of total HDL, HDL subfractions, and A-II HDL₃. Tumor necrosis factor- (TNF-) (100 U/mL) was then added to the culture medium, and 4.5 hours later the cell surface expression of VCAM-1 was measured by flow cytometry analysis.

Flow Cytometry Analysis
Levels of cell surface expression of VCAM-1 were measured as described previously.⁴ In brief, the cells were first washed with FACS wash (RPMI 1640, containing 10 mmol/L HEPES, 0.02% sodium azide, and 2.5% FCS). HUVECs were then incubated with mouse monoclonal antibody to VCAM-1 (51-10C9) for 30 minutes at 4°C and then washed again with FACS wash. Binding of the primary antibody was detected by incubation with FITC-conjugated secondary antibody [Immunotech FITC-conjugated F(ab)₂ fragment goat (mouse IgG)] for 30 minutes at 4°C. After being washed with PBS, the cells were harvested by trypsinization. FACS wash was added to neutralize the trypsin. The cells were centrifuged and the cell pellet resuspended in FACS fixative (PBS containing 2% glucose, 0.02% sodium azide, and 2.5% formaldehyde). The expression of VCAM-1 was measured as fluorescence intensity by using a Coulter Epics Profile II flow cytometer. Each sample counted 10 000 cells. Controls included preparations without the primary antibody and an isotype-matched, nonrelevant antibody.

Statistical Analyses
The data obtained in this study are expressed as mean±SEM. Students' t test for paired samples was used to determine whether differences between values were significant. Statistical significance was set at P<0.05.

	Results

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Inhibition of VCAM-1 Expression by the Total HDL Fraction
In confirmation of our earlier study,⁴ the total HDL fraction (1.07<d<1.21 g/mL) promoted a concentration-dependent inhibition of the cytokine-induced expression of VCAM-1 in endothelial cells (Figure 1). In the current study, however, the magnitude of the inhibition varied markedly between different HDL preparations. When added at equivalent concentrations of apo A-I, the HDLs isolated from subjects 1, 2, and 3 achieved much greater inhibition of VCAM-1 expression than was the case with the HDLs isolated from subjects 5 and 6. The HDLs from subject 4 were intermediate in their ability to inhibit the expression of VCAM-1. At an apo A-I concentration of 4 µmol/L, the HDLs from subjects 1, 2, and 3 inhibited VCAM-1 expression by >60%, whereas the HDLs from subjects 4, 5, and 6 inhibited expression by 38%, 16%, and 14%, respectively. These differences were apparent for apo A-I concentrations up to 16 µmol/L. Comparable intersubject variations were seen in other experiments with HDLs isolated from other subjects in studies using other preparations of HUVECs. In contrast, preparations of HDLs isolated on different occasions from the same subject did not vary in their ability to inhibit VCAM-1 expression (Figure 2).

View larger version (21K): [in this window] [in a new window]   Figure 1. Inhibition of cytokine-induced endothelial cell VCAM-1 expression by HDLs from different subjects. HUVECs were preincubated for 1 hour with HDLs isolated from each of 6 subjects before being activated with TNF- (100 U/mL) and incubated for a further 4.5 hours. Expression of VCAM-1 was quantified by flow cytometry. Values represent mean fluorescence intensity detected with antibodies directed against VCAM-1. Donors of HDLs are individually identified: subject 1 (), subject 2 (), subject 3 (), subject 4 (), subject 5 (), and subject 6 ().

View larger version (21K): [in this window] [in a new window]   Figure 2. Inhibition of cytokine-induced endothelial cell VCAM-1 expression by HDLs isolated on different occasions from individual subjects. Experiments were conducted as described in the legend to Figure 1. HDLs from each of 4 subjects were isolated on 2 occasions 3 days apart. Values are expressed as a percentage of that in a sample without HDLs. Inhibitory activities of the 2 preparations from each subject (1 to 4) are shown. Initial HDL sample () and later HDL sample () are shown for each subject.

Having found intersubject variations in the HDL-mediated inhibition of endothelial cell VCAM-1 expression, studies were next conducted to investigate whether different HDL subfractions also differed in their inhibitory activity. In the first of these we compared HDL₂ and HDL₃.

Comparison of the Inhibition of VCAM-1 Expression by HDL₂ and HDL₃
HDL₂ (1.07<d<1.12 g/mL) and HDL₃ (1.13<d<1.21 g/mL) were isolated from each of 5 subjects. Completeness of the separation of each subfraction from the other was confirmed by nondenaturing polyacrylamide gradient gel electrophoresis (Figure 3). The compositions of the HDL₂ and HDL₃ are shown in Table 1. As has been reported elsewhere,¹⁹ HDL₃s are enriched with apo A-II but depleted of cholesteryl esters, unesterified cholesterol, and triglyceride relative to HDL₂.

View larger version (22K): [in this window] [in a new window]   Figure 3. Size distributions of HDL2 and HDL3. HDL2 (1.07<d<1.12 g/mL) and HDL3 (1.13<d< 1.21 g/mL) were isolated from each of 5 subjects (1 to 5) and subjected to nondenaturing polyacrylamide gradient gel electrophoresis on 3% to 40% gels. After fixing and staining, gels were scanned with a laser densitometer. Populations of HDL2 (diameter, 10.8 to 11.6 nm) and HDL3 (diameter, 8.0 to 9.0 nm) were identified.

The HDL₂ and HDL₃ were added to HUVECs at apo A-I concentrations of 2, 4, and 8 µmol/L. Both HDL subfractions inhibited the TNF-–induced VCAM-1 expression in a concentration-dependent fashion (Figure 4). However, at all concentrations of apo A-I, the inhibition mediated by HDL₃ was greater than that of HDL₂ (Figure 4A). The differences at 4 and 8 µmol/L apo A-I were statistically significant. At 4 µmol/L apo A-I, the inhibition of VCAM-1 expression by HDL₂ and HDL₃ was 39.8±5.7% and 58.6±5.4%, respectively (P<0.05). This difference was even greater at 8 µmol/L apo A-I, with HDL₂ and HDL₃ inhibiting VCAM-1 expression by 46.2±8.0% and 71.4±5.8% respectively (P<0.05). When the inhibitory activities of HDL₂ and HDL₃ were compared in terms of their total cholesterol concentrations, the HDL₃ subfraction was still superior (Figure 4B).

View larger version (20K): [in this window] [in a new window]   Figure 4. Inhibition of endothelial cell VCAM-1 expression by HDL2 and HDL3. HUVECs were preincubated for 1 hour with preparations of either HDL2 or HDL3 shown in Figure 3 before being activated by TNF- (100 U/mL) and incubated for a further 4.5 hours. Expression of VCAM-1 was quantified by flow cytometry. Values are presented as a percentage of that in a sample without HDLs. HDL2 () and HDL3 () were added to HUVECs according to apo A-I concentration (A) and total cholesterol concentration (B). Results are mean and SEM of results with HDL subfractions isolated from each of 5 subjects. Differences between means were evaluated by paired t tests. *P<0.05.

Effect of Removing HDL₂ and HDL₃ Prior to Adding TNF-
To determine whether the difference between HDL₂ and HDL₃ was dependent on the HDL subfractions' being physically present at the time the TNF- was added, studies were conducted with cells that had been preincubated for 2 hours with each of the HDL subfractions. The HDL subfractions were then removed before TNF- was added. Before the TNF- was added, the cells were washed twice with fresh medium to ensure maximal removal of the HDLs. With this technique, >95% of the added HDL (expressed as apo A-I) was accounted for in the removed medium.

Despite removal of the HDLs before the addition of TNF-, expression of VCAM-1 was still inhibited in a concentration-dependent manner (Figure 5). Furthermore, the superior inhibitory activity of HDL₃ persisted. At concentrations of apo A-I of 2, 4, and 8 µmol/L during preincubation, the subsequent inhibition of TNF-–induced VCAM-1 expression was, respectively, 14%, 32%, and 46% in the case of HDL₂ and 31%, 50%, and 60% in the case of HDL₃.

View larger version (15K): [in this window] [in a new window]   Figure 5. Effect of HDL2 and HDL3 when removed from HUVECs before addition of cytokine. HUVECs were preincubated for 2 hours with either HDL2 or HDL3. Medium containing HDLs was then removed and cells were washed twice with fresh medium. Cells were then stimulated with cytokine TNF- (100 U/mL) and the incubation continued for an additional 4.5 hours. Cells were then assayed for cell surface VCAM-1 expression by flow cytometry. HDL2 () and HDL3 () were added to cells according to apo A-I concentration. Results are mean and SEM from 3 experiments, each performed in duplicate.

Effects of Isolated HDL₂ and HDL₃ Apolipoproteins and Lipids on VCAM-1 Expression
To investigate the possibility that the differences between HDL₂ and HDL₃ may have reflected differences in their protein or lipid constituents, their apolipoproteins and lipids were isolated and their effects on TNF-–induced VCAM-1 expression determined. In marked contrast to the intact HDL subfractions, neither the apolipoproteins (Figure 6A) nor sonicated preparations of the lipids (Figure 6B) from either subfraction had any significant inhibitory activity. Similarly, when HUVECs were incubated with purified apo A-I (up to 24 µmol/L) in the absence of lipid or with sonicated, small, unilamellar vesicles prepared with POPC (up to 3.2 mmol/L) in the absence of apolipoproteins, there was no inhibition of the TNF-–induced expression of VCAM-1 (result not shown). When, however, apo A-I and POPC were reconstituted into discoidal rHDLs, the resulting complexes promoted a concentration-dependent inhibition of VCAM-1 expression comparable to that seen with native HDLs (Figure 7).

View larger version (21K): [in this window] [in a new window]   Figure 6. Inhibitory activities of lipid-free apolipoproteins and extracted lipids isolated from HDL2 and HDL3. HUVECs were preincubated for 1 hour with either the isolated apolipoproteins of HDL2 or HDL3 (A) or the extracted lipids of HDL2 or HDL3 (B) before being activated with TNF- and assayed for cell surface VCAM-1 expression as described in the legend to Figure 1. Values are expressed as a percentage of that in a sample without HDLs. Proteins from HDL2 () and HDL3 () were added to HUVECs according to apo A-I concentration. Lipid components of HDL2 () and HDL3 () were added to the HUVECs according to total cholesterol concentration. Results are mean of 2 separate experiments, each performed in duplicate.

View larger version (15K): [in this window] [in a new window]   Figure 7. Inhibitory activity of rHDLs. Discoidal rHDLs consisting of complexes of apoA-I and POPC were incubated with HUVECs as described in the legend to Figure 1. Cells were then activated with TNF- and assayed for VCAM-1 expression as outlined in the legend to Figure 1. Values are expressed as a percentage of that in a sample without HDLs. Results are mean and SEM from 6 experiments, each performed in duplicate.

Effect of Apolipoprotein Composition on the Inhibition of VCAM-1 Expression by HDL₃
HDL₃ includes 2 main apolipoprotein-specific subpopulations: 1 containing apo A-I only (A-I HDL₃) and the other containing both apo A-I and apo A-II (A-I/A-II HDL₃).²⁰ To determine whether changing the apolipoprotein composition of HDL₃ impacts the ability of these lipoproteins to inhibit VCAM-1 expression, preparations of HDL₃ were modified by replacing all of their apo A-I with apo A-II to form (A-II) HDL₃. This replacement of apo A-I with apo A-II increased the size of the HDL₃ particles slightly (from 8.9 to 9.2 nm, Figure 8B) but did not change their lipid composition (Table 2). The modified HDL₃s containing only apo A-II were then compared with the unmodified particles, which comprised a mixture of (A-I) and (A-I/A-II) HDL₃ (Figure 8A).

View larger version (20K): [in this window] [in a new window]   Figure 8. Comparison of inhibitory activities of unmodified HDL3 and A-II HDL3. HUVECs were preincubated with unmodified HDL3 or A-II HDL3 (prepared as described in Methods) for 1 hour and then stimulated with TNF- (100 U/mL). Incubation was then continued for an additional 4.5 hours before cells were assayed for cell surface VCAM-1 expression by flow cytometry. Values are expressed as a percentage of that in a sample without HDLs. Unmodified HDL3 () and A-II HDL3 () were added to HUVECs according to total cholesterol concentration (A). Results are mean and SEM from 3 experiments, each performed in duplicate. Unmodified HDL3 and A-II HDL3 were also subjected to nondenaturing polyacrylamide gradient gel electrophoresis as outlined in the legend to Figure 2 (B). A-II HDL3 and unmodified HDL3 had Stokes' diameters of 9.2 and 8.9 nm, respectively. Profiles in B are from a single experiment but are representative of results from 3 experiments.

Both the unmodified HDL₃ and (A-II) HDL₃ inhibited the cytokine-induced expression of VCAM-1 in endothelial cells in a concentration-dependent fashion (Figure 8A). When equated for cholesterol concentration (and thus for HDL particle concentration), there was no difference in the magnitude of the inhibition achieved by the 2 preparations.

	Discussion

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These studies show that HDLs isolated from different subjects may vary markedly in their ability to inhibit the cytokine-induced expression of VCAM-1 in endothelial cells. They also show that the inhibitory activity of HDL₃ is substantially greater than that of HDL₂.

The apparent superiority of HDL₃ over HDL₂ in terms of inhibiting VCAM-1 expression may be a consequence of differences in either the particle size or the composition of the subfractions. It is well known that other functions of HDLs, such as their interaction with lecithin:cholesterol acyltransferase²¹ and hepatic lipase,²² are influenced by variations in both of these parameters. Other more subtle differences between the HDL subfractions, such as variations in the conformation of their apolipoproteins, may also impact on function by altering the surface exposure of specific apolipoprotein epitopes. It is possible, however, that the superiority of HDL₃ as an inhibitor of VCAM-1 is unrelated to any of these differences in size, composition, or structure. Rather, it may be a simple reflection of the fact that when the 2 subfractions are equated for concentrations of either apo A-I or cholesterol, there are more HDL₃ particles than HDL₂ particles. This issue is being addressed in studies using rHDLs in which the size and composition can be more tightly controlled.

The mechanism by which HDLs inhibit endothelial cell adhesion molecule expression is uncertain. One possibility relates to the ability of HDLs to function as antioxidants. The fact that endothelial cell VCAM-1 expression may be induced by redox-sensitive transcriptional events²³ raises the possibility that the ability of HDLs to inhibit endothelial cell adhesion molecule expression may be secondary to their known capacity to remove any lipid oxidation products²⁴ that may be generated by TNF-. If this were so, it could be argued that the difference between the inhibitory activities of HDL₂ and HDL₃ may reflect their relative abilities to remove such lipid oxidation products. However, this mechanism was not consistent with the observation that the inhibitory activities of the HDL subfractions remained different even when the HDLs were present only during preincubations and were removed from the endothelial cells before the addition of TNF- (Figure 5). These preincubation studies also excluded the possibility that the HDLs may have acted by interfering with the binding of TNF- to its cell receptors. Rather, it appears that HDLs somehow "prime" endothelial cells to be resistant to the cytokine-induced expression of VCAM-1.

Insights into which components of HDLs are responsible for the inhibition of VCAM-1 expression were obtained from the studies with discoidal rHDLs (Figure 7). Discoidal complexes containing only apo A-I and POPC were able to mimic native HDLs in their ability to inhibit endothelial cell VCAM-1 expression. The fact that neither lipid-free apoA-I alone nor POPC vesicles that did not contain apo A-I had inhibitory activity indicated that both apolipoproteins and lipids are required for the effect. In retrospect, it was therefore predictable that there was no inhibitory activity detected in either the delipidated proteins or the extracted lipids isolated from HDL₂ and HDL₃. This negative result frustrated attempts to determine whether the differing inhibitory activities of the 2 HDL subfractions resided in their lipid or apolipoprotein moieties. However, it was possible to show that major changes to the apolipoprotein composition of HDL₃ had little effect on its ability to inhibit endothelial cell VCAM-1 expression. This observation is consistent with a recent report that rHDLs containing apoA-I are comparable to those containing apoA-II as inhibitors of endothelial cell VCAM-1 expression,²⁵ although it does not address the possibility of differences between A-I HDLs and A-I/A-II HDLs, the 2 main apolipoprotein-specific HDL subpopulations in human plasma.

The relationship between the inhibition of endothelial cell adhesion molecule expression and the antiatherogenic properties of HDL subpopulations is not known. This is not a trivial issue, because human and animal studies have suggested that HDL₂ may differ from HDL₃ and A-I HDLs may differ from A-I/A-II HDLs in terms of their cardioprotective properties. Several reports have concluded that HDL₂s are superior to HDL₃s,^{26 27 28} although there have also been conclusions that HDL₃ may be the superior antiatherogenic subfraction.^{29 30 31} The observation that high levels of HDL cholesterol are generally indicative of high levels of HDL₂ highlights the importance of HDL₂ as a cardioprotective fraction. However, the probability that HDL₃ is also cardioprotective is suggested by the fact that an inverse relationship between the concentration of HDL cholesterol and the risk of developing coronary heart disease persists even when the total HDL cholesterol concentration is <1.0 mmol/L and there is no measurable HDL₂.

There is also evidence that apolipoprotein composition may be an important determinant of the ability of HDLs to inhibit atherosclerosis. Studies of transgenic animals^{32 33 34} suggest that A-I HDLs may be superior to both A-I/A-II HDLs and A-II HDLs in their ability to protect against atherosclerosis. However, as with HDL₂ and HDL₃, the situation in humans is uncertain, with circumstantial evidence both in favor³⁵ and against³⁶ the proposition that A-I HDLs account for most of the ability of HDLs to protect against coronary heart disease. It remains to be determined whether A-I HDLs are also superior to A-I/A-II HDLs in their ability to inhibit endothelial cell adhesion molecule expression and, if so, whether such superiority translates into an enhanced ability to inhibit the development of atherosclerosis.

In conclusion, the ability of HDLs to inhibit the cytokine-induced expression of endothelial cell VCAM-1 has been confirmed and extended by finding marked differences in the inhibitory activities of the 2 major HDL subfractions and in HDL preparations isolated from different human subjects. The mechanism responsible for these differences is not immediately apparent. Future studies designed to investigate whether this inhibition of endothelial cell adhesion molecule expression contributes to the ability of HDLs to inhibit the development of atherosclerosis are awaited with interest.

	Acknowledgments

 
This work was supported by funding from the National Health and Medical Council of Australia (to P.J.B.), the National Heart Foundation of Australia (to P.J.B.), and the Royal Adelaide Hospital Research Fund (to P.J.B.). D.T.A. was supported by a fellowship from the Helpman Bequest. We thank Jenny Drew for technical assistance in the growth of endothelial cells. TNF- was a generous gift of Genentech, South San Francisco, Calif. We also thank the staff of the delivery wards of the Women's and Children's Hospital and Burnside War Memorial Hospital, Adelaide, for the collection of umbilical cords.

Received July 16, 1997; accepted March 27, 1998.

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