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Originally published In Press as doi:10.1194/jlr.M400229-JLR200 on August 16, 2004

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Journal of Lipid Research, Vol. 45, 2116-2122, November 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation

Po-Yuan Chang1,*, Shao-Chun Lu1,{dagger}, Ta-Chen Su*, San-Fang Chou{dagger}, Wen-Huei Huang{dagger}, Joel D. Morrisett§, Chu-Huang Chen§, Chiau-Suong Liau* and Yuan-Teh Lee2,*

* Departments of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
{dagger} Biochemistry and Molecular Biology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
§ Department of Medicine, Baylor College of Medicine, Houston, TX

Published, JLR Papers in Press, August 16, 2004. DOI 10.1194/jlr.M400229-JLR200

1 P-Y. Chang and S-C. Lu contributed equally to this study. Back

2 To whom correspondence should be addressed. e-mail: ytlee{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypercholesterolemic human LDL contains oxidized subfractions that have atherogenic properties. Paradoxically, atherosclerosis incidence is low in patients with primary biliary cirrhosis (PBC), a disease characterized by marked increases in plasma LDL, including the LDL subfraction lipoprotein-X (Lp-X). To investigate the mechanisms underlying this paradox, we first examined the propensity to oxidation of unfractionated LDL isolated from PBC patients. After prolonged incubation with copper, PBC-LDL failed to increase the oxidation index or electrophoretic mobility noted in control LDL. An admixture of PBC-LDL or Lp-X with control LDL prevented oxidation of the latter in a dose-dependent manner. PBC-LDL was also noncompetitive against copper-oxidized LDL (oxLDL) for binding with a murine monoclonal anti-oxLDL antibody in a competitive ELISA. OxLDL exerts its proapoptotic and antiangiogenic effects in part by inhibiting fibroblast growth factor 2 (FGF2) expression. Preincubation of oxLDL with PBC-LDL, but not control LDL, attenuated the inhibitory effects of oxLDL on FGF2 expression in cultured bovine aortic endothelial cells (ECs). The antioxidant and prosurvival properties of PBC-LDL diminished after the patients underwent orthotopic liver transplantation.

These results suggest that Lp-X reduces LDL atherogenicity by preventing LDL oxidation to protect EC integrity in the presence of hypercholesterolemia. They also suggest that altering LDL composition may be as important as reducing LDL concentration in preventing or treating atherosclerosis.

Abbreviations: BAEC, bovine aortic endothelial cell; EC, endothelial cell; FGF2, fibroblast growth factor 2; LDL-C, low density lipoprotein cholesterol; Lp-X, lipoprotein-X; oxLDL, copper-oxidized low density lipoprotein; PBC, primary biliary cirrhosis; TBARS, thiobarbituric acid-reactive substances

Supplementary key words hypercholesterolemia • low density lipoprotein oxidation • fibroblast growth factor 2 • endothelium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypercholesterolemia increases atherosclerosis incidence in the general population but not in patients with primary biliary cirrhosis (PBC), a cholestatic liver disease often associated with marked increases in plasma LDL cholesterol (LDL-C) (1–3). Despite the highly increased cholesterol levels, prospective observation for a median of 7.4 years of 312 patients with PBC of various stages found no increased incidence of atherosclerotic death compared with age- and sex-matched controls (4). Factors that may account for this apparent paradox are not clearly defined. Lipoprotein-X (Lp-X), an LDL rich in phospholipid and nonesterified cholesterol but poor in cholesteryl ester, triglyceride, and protein, is characteristic of advanced PBC (3). In all three isoforms, Lp-X1, Lp-X2, and Lp-X3, the particles are rich in phospholipids (65–67%) and cholesterol (23–27%) but poor in cholesteryl esters (0.5–2%), triglycerides (2–3%), and protein (3–7%), giving them a hydrated density similar to that of LDL (5). The lack of association between high LDL-C and atherosclerosis in PBC raises the question of the atherogenic properties of PBC-LDL and prompted us to investigate the physiological relevance of Lp-X in atherogenesis.

The bioactivities of oxidized products of LDL may account for LDL atherogenicity (6). If PBC-LDL is resistant to oxidation, a lesser amount of such oxidized metabolites will be accumulated. Therefore, we first assessed the susceptibility of PBC-LDL to oxidation. Because PBC-LDL may contain a variable percentage of Lp-X, we also tested whether Lp-X renders LDL resistant to oxidants. Fibroblast growth factor 2 (FGF2), by functioning as an autocrine and paracrine agent, regulates the proliferation and apoptosis of vascular endothelial cells (ECs) by activating the phosphatidylinositol 3-kinase-protein kinase Akt pathway (7, 8). Both copper-oxidized LDL (oxLDL) and hypercholesterolemic electronegative LDL inhibit proliferation and induce apoptosis in ECs in part by downregulating expression of the FGF2 gene (9–11). Whether PBC-LDL regulates FGF2-mediated survival mechanisms depends on its effect on FGF2 expression. This important issue was also addressed in this study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
EDTA was from Becton-Dickinson (Rutherford, NJ). The Superose 6HR 10/30 column was from Pharmacia LKB (Uppsala, Sweden). Enzymatic kits for cholesterol and triglyceride analyses were from E. Merck (Darmstadt, Germany). The Beckman Paragon system was from Beckman (Palo Alto, CA). DMEM was from Invitrogen (Carlsbad, CA). Nonidet P-40 was from Sigma (St. Louis, MO).

LDL, Lp-X, and oxLDL preparation
LDL (d = 1.019–1.063 g/ml) was isolated by sequential ultracentrifugation from the plasma of six PBC patients after fasting (Table 1). To isolate Lp-X, PBC-LDL and LDL obtained from healthy, normolipidemic subjects were subjected to gel filtration chromatography using a Superose 6HR 10/30 column at 0.25 ml/min and collected in 0.5 ml fractions (12). OxLDL was prepared by incubating control LDL with 10 µM CuSO4 for 24 h as previously described (13). Protein in LDL preparations was estimated by the Lowry method (14), and thiobarbituric acid-reactive substances (TBARS) were assayed as a measure of oxidative lipid modification (13). Cholesterol and triglyceride concentrations in plasma and LDL preparations were determined enzymatically as previously described (15).


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  		TABLE 1. Characteristics and antioxidation index in six patients with primary biliary cirrhosis

 
Oxidation and oxidizability analyses
The oxidizability of LDL preparations was determined by their electrophoretic mobility after being exposed to 10 µM CuSO4 at 37°C for 1–18 h on agarose gels using the Beckman Paragon system (16). A competitive ELISA was established using an oxLDL-specific monoclonal antibody, mAb-4E6, a generous gift from Professor Paul Holvoet at the Center for Experimental Surgery and Anesthesiology, Leuven, Belgium (17). In brief, oxLDL (1 µg/ml, 100 µl/well) was used as the plated antigen. mAb-4E6 was added in the presence or absence of competing ligands: oxLDL, PBC-LDL, and control LDL. Samples were incubated for 2 h at room temperature. After washing, the wells were incubated for 1 h with horseradish peroxidase-conjugated rabbit IgG raised against mouse immunoglobulins and washed again. The peroxidase reaction was performed as previously described (17), and the absorbance was read at 492 nm.

Cell culture
Primary cultures of bovine aortic endothelial cells (BAECs) were derived and purified as previously described (18). Cells at 8–12 passages, maintained in DMEM supplemented with 10% FBS and antibiotics (100 µg/ml streptomycin, 100 IU/ml penicillin, and 0.25 µg/ml amphotericin B), were used. Cell cultures grown to subconfluence were washed three times with serum-free medium and maintained under serum-free conditions for 6 h before being treated with PBS (lipoprotein-free control), control LDL (50 µg/ml), oxLDL (50 µg/ml), or PBC-LDL (50 µg/ml) for 24 h. For the study of DNA synthesis and intracellular FGF2 protein, 1 x 106 cells were seeded in each well of 12-well Corning cell culture plates.

Determination of intracellular FGF2 protein and mRNA levels
Intracellular FGF2 concentrations in cell lysates prepared with Nonidet P-40 were measured by ELISA using a Quantikine kit. As previously described (18), FGF2 concentrations were estimated at 450 nm spectrophotometrically using standard curves. FGF2 mRNA levels were determined by RT-PCR; 1 µg of the extracted total RNA was reverse transcribed in 10 µl of reaction mixture containing 2.5 units of Moloney murine leukemia virus reverse transcriptase for 45 min at 42°C. RT-PCR was performed according to procedures described previously (10). The FGF2 primers were 5'-GGAGTGTGTGCTAACCGTTACCTGGCTATG-3' (upstream) and 5'-TCAGCTCTTAGCAGACATTGGAAGAAAAAG-3' (downstream). As an internal control, ß-actin was used with the following primers: 5'-AACCGCGAGAAGATGACCCAGATCATGTTT-3' (upstream) and 5'-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3' (downstream). A fraction of each PCR product (10 µl) was electrophoresed on a 2% agarose gel, and DNA bands stained with ethidium bromide were visualized by UV transillumination.

Statistical analysis
The significance of the differences between group means was assessed by a two-sided Student's t-test for single comparisons and by Bonferroni's test for multiple comparisons. Probability values of <0.05 were considered significant. Results are expressed as means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Of the six PBC patients examined, two were Scheuer stage III, three were stage II, and one was stage I. The percentage of LDL present as Lp-X increased with the severity of the disease and was as high as 90% of total LDL in the stage III patients (Table 1). Because the bioactivity of PBC-LDL depends on its content of Lp-X (see below), we describe the functional properties of PBC-LDL obtained from the patient with the highest Lp-X content. The patient was a 47 year old woman hospitalized in January 1999 for evaluation of intractable pruritus and generalized xanthomas. Serum values included antimitochondrial antibody titer of 1:160, alkaline phosphatase of 2,244 units/l, {gamma}-glutamyl transpeptidase of 1,040 units/l, total bilirubin of 222.3 µmol/l, total cholesterol of 34.1 mmol/l, and LDL-C of 31.7 mmol/l. Hepatobiliary scintigraphy and liver biopsy results were compatible with Scheuer stage III PBC. Despite marked hypercholesterolemia and widely distributed xanthomas, coronary angiographic and carotid ultrasonographic findings were normal. The patient denied symptoms of chest pain or intermittent claudication. A treadmill test was normal. Analysis of the patient's LDL by Superose 6HR 10/30 column chromatography showed that 90% of the patient's total LDL was accounted for by Lp-X, seen as an additional peak eluting in fractions 17–20 compared with a single peak in fractions 23–29 for control LDL (Fig. 1). None of the other five patients had a history of chest angina or peripheral symptoms that were ischemia related.



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  		Fig. 1. Gel filtration chromatography of unfractionated LDL from plasma of a patient and a healthy subject (control). Lipoproteins of 1.019 < d < 1.063 isolated from 1 ml of plasma were applied to a Superose 6HR 10/30 column at a flow rate of 0.25 ml/min and collected 0.5 ml/tube. The fractions of lipoprotein-X (Lp-X) and normal LDL are indicated. Experiments using primary biliary cirrhosis (PBC)-LDL samples obtained from the other five patients yielded similar results to varying degrees, depending on the Lp-X content.

 
Exposure of the patient's total LDL fraction to copper for up to 18 h failed to induce a change in mobility on agarose gel electrophoresis (Fig. 2A), and TBARS remained <1 nmol/mg LDL protein (data not shown). In contrast, LDL isolated from a healthy subject was readily oxidized, exhibiting movement toward the anode by 2 h (Fig. 2A); TBARS concentration increased to 16 nmol/mg at 18 h. Equal-concentration (200 µg/ml) mixing with the patient's unfractionated LDL suppressed control LDL oxidizability and completely prevented a change in control LDL mobility (Fig. 2B). The extent of inhibition in electrophoretic mobility by PBC-LDL correlated well with the PBC-LDL molar ratio to control LDL. As shown in Fig. 3, an admixture of PBC-LDL and control LDL at 1:9, 1:4, or 1:1 exhibited a dose-response effect on the mobility change after exposure to copper for 8 h (Fig. 3). To determine the antioxidant properties of the subfractions of PBC-LDL, electrophoretic mobility was assessed in the Lp-X (fractions 17–20) and the normal LDL (fractions 23–29) from the same patient. At 2 h of copper exposure, the patient's Lp-X was resistant to oxidation, whereas her normal LDL exhibited electrophoretic mobility changes proportional to the duration of exposure (Fig. 4A). Her normal LDL fraction became completely oxidation resistant after admixture with an equal amount of her Lp-X (Fig. 4B). Using the ability of PBC-LDL to attenuate the electrophoretic mobility as an index, we found that such an ability depended on the content of Lp-X (Table 1). In both Scheuer stage II patients, the PBC-LDL had a marked antioxidant effect, which was negligible in PBC-LDL with minimal Lp-X. Of note, although plasma concentrations of bilirubin, a potent antioxidant, increased as Lp-X levels increased (Table 1), bilirubin was not detected in Lp-X.



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  		Fig. 2. LDL oxidation assessed by agarose gel electrophoresis of lipoproteins from a patient with stage III PBC and a healthy subject (control). A: Patient and control unfractionated LDLs (200 µg/ml) were incubated with 10 µM CuSO4 for 0–18 h as indicated. B: Equal-concentration mixing of patient and control unfractionated LDLs. The assay procedures are described in the text. Experiments using PBC-LDL samples obtained from the other five patients yielded similar results to varying degrees, depending on the Lp-X content.

 


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  		Fig. 3. Dose-dependent effect of patient LDL on LDL oxidation. Patient and control unfractionated LDLs were mixed at 1:9, 1:4, or 1:1 ratio as indicated and incubated with 10 µM CuSO4 for 8 h. LDL oxidation was assessed by agarose gel electrophoresis. Experiments using PBC-LDL samples obtained from other patients yielded similar results to varying degrees, depending on the Lp-X content.

 


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  		Fig. 4. Oxidation analysis of isolated Lp-X and LDL of a PBC patient. A: Patient Lp-X (fractions 17–20) and normal LDL (fractions 23–29) were incubated with 10 µM CuSO4 for 0–2 h as indicated followed by agarose gel electrophoresis. B: Equal-concentration mixing of patient Lp-X and normal LDL fractions incubated with 10 µM CuSO4 for 0–2 h as indicated.

 
The oxidizability of patient LDL was also assessed by competitive ELISA using the anti-oxLDL monoclonal antibody mAb-4E6. Copper-treated patient LDL at more than 1,000-fold higher molar concentrations failed to compete with oxLDL for binding (Fig. 5). The binding capability of patient LDL was comparable to that of control LDL, confirming an oxidation-resistant feature of Lp-X.



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  		Fig. 5. Evaluation of the oxidizability of LDL by ELISA. A competitive ELISA was used to measure the oxidizability of LDL using the copper-oxidized LDL (oxLDL)-specific monoclonal antibody mAb-4E6 according to the protocols described in the text. Competing ligands included oxLDL, copper-treated PBC-LDL (Cu-PBC-LDL), and control LDL as indicated. B and B0 represent absorbance in the presence and absence of competitor, respectively. Experiments using PBC-LDL samples obtained from the other five patients yielded similar results to varying degrees, depending on the Lp-X content.

 
Endothelial survival was measured by DNA synthesis and FGF2 expression in cultured BAECs. Treatment of BAECs with oxLDL produced concomitant reductions in intracellular FGF2 peptide levels and cell proliferation. Unlike control LDL (50 µg/ml), PBC-LDL (50 µg/ml) incubated with copper for up to 24 h failed to reduce the FGF2 protein concentration. Preincubation of oxLDL (50 µg/ml) with PBC-LDL attenuated FGF2 reduction in a dose-dependent manner, whereas preincubation with control LDL did not alter the inhibitory effect of oxLDL (Fig. 6A). The FGF2 mRNA level was also reduced by oxLDL but not by PBC-LDL. Preincubation of oxLDL with PBC-LDL also attenuated oxLDL-induced FGF2 mRNA downregulation (Fig. 6B). In accordance with the changes in FGF2 expression, oxLDL decreased DNA synthesis by half, whereas PBC-LDL had no effect. Preincubation of oxLDL with PBC-LDL, but not control LDL, prevented the inhibitory effect of oxLDL (data not shown).



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  		Fig. 6. Effects of LDL on intracellular fibroblast growth factor 2 (FGF2) expression. A: FGF2 peptide levels measured by ELISA. Bovine aortic endothelial cells (BAECs) were incubated with different LDL preparations (50 µg/ml each) as indicated for 24 h before ELISA, according to the protocols described in the text. Values shown are means ± SEM (n = 3). Each well contained 1 x 106 cells at inoculation. * P < 0.05 vs. PBS control. B: FGF2 mRNA measured by RT-PCR. BAECs were incubated with different LDL preparations (50 µg/ml each) as indicated for 24 h, and isolated total RNA was subjected to RT-PCR analysis as described in the text. Data are representative of three separate experiments with similar results.

 
After the patient received an orthotopic liver transplant in March 1999, her total cholesterol decreased within 2 weeks to 2.6 mmol/l, Lp-X disappeared, and the LDL no longer exerted antioxidant action (data not shown). In contrast to her pretransplantation LDL, which failed to induce a change in mobility after copper oxidation (Fig. 2A), her posttransplantation LDL exhibited electrophoretic mobility changes proportional to the duration of copper exposure. The oxidizability was not different from that of control LDL. Admixing control LDL with patient LDL no longer attenuated the mobility of control LDL (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hypercholesterolemic human plasma contains atherogenic LDL subfractions that can inhibit proliferation and increase apoptosis in ECs and induce monocyte-EC adhesion (9, 19). However, despite the highly increased cholesterol levels and marked xanthomas commonly found in patients with PBC (2, 3), the incidence of atherosclerotic cardiovascular risk does not increase in these patients (4). Our findings indicate that LDL isolated from the plasma of PBC patients not only protected normolipidemic LDL from becoming oxidized but also prevented oxLDL from disrupting the FGF2-dependent survival mechanism in vascular ECs. The antioxidant effect of PBC-LDL depended on its Lp-X content. These findings may further substantiate the LDL oxidation hypothesis for the pathogenesis of atherosclerosis.

The atherogenic properties of LDL have been postulated to result from oxidative modification of the lipoprotein (6, 20–22). Clinically, the incidence of acute coronary events is related to the concentration of circulating oxLDL detectable by immunoassay (23, 24). Electronegative LDL is a naturally occurring, mildly oxidized LDL species that can induce a variety of atherogenic effects in vascular ECs (9, 19, 25). Because electronegative LDL is more abundant in hypercholesterolemic than normolipidemic human plasma (9), hypercholesterolemic LDL should have a greater susceptibility to oxidation than normolipidemic LDL. Failure of hypercholesterolemia to increase atherosclerosis incidence in PBC raised the question of whether PBC-LDL is resistant to oxidative modification.

Lipid peroxidation-producing products, including 4-hydroxynonenal, are considered important in the pathologic process leading to the bile duct damage of PBC (26). In early PBC, lipid peroxidation markers, including plasma and urinary 8-isoprostane and plasma malondialdehyde, are increased, whereas plasma antioxidants, such as total glutathione, are decreased (27). Although these changes lead to eventual bile duct pathology, their effects on the vascular wall have not been documented. In this report, severe biliary cirrhosis and extensive occurrence of xanthomas were not accompanied by coronary or carotid narrowing.

Previous reports have attributed the paradoxically low atherosclerosis incidence in PBC to high plasma concentrations of HDL and Lp-A-I (28) and the observation that most patients were premenopausal women (2). Although the contribution of these protective factors cannot be discounted, premature coronary disease in patients with LDL-C increased to the degree seen in PBC patients is well documented in non-PBC patients regardless of their overall lipid profile, age, or gender. Our data show that PBC-LDL was resistant to copper oxidation, probably because of the high rigidity of the particle, thereby preventing penetration of copper ions into the hydrophobic region of the bilayer, where oxidizable unsaturation sites are localized. Even more important, the Lp-X fraction isolated from PBC-LDL rendered the non-Lp-X fraction from the same origin resistant to copper oxidation. This may have been attributable to the higher affinity of Lp-X than LDL for copper ions. The capability of PBC-LDL to suppress oxidation-induced change in the electrophoretic mobility of control LDL appeared to reflect the Lp-X/LDL concentration ratio. These findings provide a new explanation for vascular protection in PBC other than the HDL, age, and gender parameters. Bilirubin, a potent physiological antioxidant in plasma, has been shown to inhibit LDL oxidation in vitro (29). The concomitant increase in plasma bilirubin with Lp-X may play an additional role in shielding LDL from oxidation in patients with PBC. However, the antioxidant effect of Lp-X observed in this study was independent of bilirubin, because there was a lack of detectable bilirubin in these Lp-X fractions.

In this study, copper ion was used as an initiating agent for LDL oxidation. Copper-mediated LDL oxidation is the most common means of initiating LDL oxidation in vitro (30), and the copper-catalyzed processes are of potential physiological relevance. Studies have shown that oxidized products of LDL induced by copper are similar to those occurring in the presence of cells (13), and the biological properties of oxLDL are very similar to those of atherogenic LDL isolated from hypercholesterolemic human plasma (9, 19). Furthermore, atheromatous lesions have been shown to contain copper ion in forms that can catalyze free radical formation (31). The role of copper ion as a catalyst in LDL oxidation may be limited by existing metal-chelating agents such as thiols that can inhibit copper-dependent LDL oxidation in vivo (32, 33). In our study, however, it is unlikely that the antioxidant activity of Lp-X was caused by copper ion chelation, because Lp-X lost its antioxidant activity gradually when unfractionated patient LDL was stored at 4°C and during gel-filtration purification of Lp-X (data not shown).

FGF2 is a pleiotropic protein that exerts angiogenic and antiapoptotic effects by activating the phosphatidylinositol 3-kinase-Akt system and the subsequent signaling pathways (7, 8). Both oxLDL and electronegative LDL downregulate FGF2 expression in ECs, contributing to reduced proliferation and increased apoptosis (9–11). In this study, PBC-LDL did not inhibit FGF2 expression or reduce DNA synthesis in BAECs. Moreover, it completely prevented the inhibitory effects of oxLDL on these parameters. This attenuation cannot be explained by dilution of oxLDL after 1:1 admixture, because control LDL failed to attenuate oxLDL's effects after a similar admixture.

These findings indicate that PBC-LDL protected ECs in two ways. First, it prevented the oxidation of normal LDL, as confirmed by Lp-X's protection of the Lp-X-free part of LDL from the same individual against oxidation. Second, PBC-LDL effectively attenuated the adverse effects of LDL that had already been oxidized on ECs. The second protective mechanism is not clear at this time. Because hydrolyzing platelet-activating factor or platelet-activating factor-like lipids accumulated in oxLDL abolishes the bioactivities of the lipoprotein (11), one possibility is that PBC-LDL or Lp-X is rich in phospholipases that can degrade these lipids in oxLDL. Further experiments are required to test this possibility.

The striking disappearance of the protection of the PBC patient's LDL from oxidation after liver transplantation further confirmed the antioxidant role of PBC-LDL attributable to its Lp-X components. These results are not consistent with the view that the low incidence of atherosclerosis in PBC is primarily a consequence of concomitant increases in HDL and Lp-A-I.

In conclusion, our findings indicate that PBC-LDL protects EC integrity by preventing LDL oxidation and the adverse effects of LDL with any degree of oxidative modification that may have occurred in vivo. Although the mechanism is not completely elucidated at this time, the antioxidant effect of PBC-LDL can be attributed to its Lp-X component. The findings also suggest that changing the composition of LDL to render it resistant to oxidants may be as important as decreasing the plasma concentration of LDL in the prevention or treatment of atherosclerosis.


   ACKNOWLEDGMENTS
 
This study was supported by Grants NSC 89-2316-B-002-017-M52 and NSC 91-2320-B-002-185 from the National Science Council, Taipei, Taiwan (P-Y.C., Y-T.L., S-C.L.), by Grants NTUH.92A14 and 93A02 from National Taiwan University Hospital, Taipei, Taiwan (P-Y.C.), and by Research Grant 1-04-RA-13 from the American Diabetes Association (C-H.C.). The authors are grateful to Kerrie C. Jara for editorial assistance.

Manuscript received June 15, 2004 and in revised form July 26, 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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