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Originally published In Press as doi:10.1074/jbc.M403271200 on April 27, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28662-28669, July 2, 2004
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The Peroxisome Proliferator-activated Receptor {alpha} (PPAR{alpha}) Agonist Ciprofibrate Inhibits Apolipoprotein B mRNA Editing in Low Density Lipoprotein Receptor-deficient Mice

EFFECTS ON PLASMA LIPOPROTEINS AND THE DEVELOPMENT OF ATHEROSCLEROTIC LESIONS*

Tao Fu{ddagger}, Debnath Mukhopadhyay§, Nicholas O. Davidson§, and Jayme Borensztajn{ddagger}

From the {ddagger}Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611 and the §Department of Internal Medicine, Washington University, St. Louis, Missouri 63110

Received for publication, March 24, 2004 , and in revised form, April 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein B (apoB)1 occurs in two forms, apoB-100 and apoB-48 (1). ApoB-100 is essential for the assembly of VLDL in the liver, and apoB-48 is essential for the assembly of chylomicrons in the intestine. In rodents, apoB-48 is also synthesized in the liver and secreted into the circulation associated with VLDL (1). Both apoBs are the products of a single gene. ApoB-48 is synthesized as a result of apoB mRNA editing, a process that requires a multicomponent enzyme complex containing an RNA-specific cytidine deaminase, apobec-1, and an RNA-binding subunit, apobec-1 complementation factor (ACF) (2).

In addition to its essential role in the assembly of VLDL in the liver, apoB-100, but not its edited form apoB-48, also plays an important role in the removal of these lipoproteins from circulation (3). This removal is a multistep process that begins when the VLDL secreted into the circulation undergo partial degradation by lipoprotein lipase present on the surface of endothelial cells, giving rise to cholesterol-enriched remnant particles of intermediate and low densities, the IDL and LDL. The remnants are then transported to the liver where they are endocytosed by hepatocytes. In normal animals, the endocytosis of the remnants is mediated primarily by the low density lipoprotein receptor, which binds apoB-100, as well as apoE, with high affinity. In animals that lack the LDLR (e.g. homozygous LDLR knock-out mice), remnants are endocytosed by hepatocytes through the mediation of the low density lipoprotein receptor-like protein (LRP), which recognizes apoE but not apoB-100 or apoB-48 (3). However, LRP alone cannot efficiently handle the removal of remnants from the plasma when the LDLR is absent, even when the influx of VLDL into the circulation is normal, as when the animals are fed a regular rodent low fat chow diet. As a result, LDLR-deficient mice accumulate apoB-100- and apoB-48-carrying remnants in the plasma, giving rise to a characteristic mild hypercholesterolemia (4). It might be anticipated, therefore, that reducing the secretion of VLDL by the liver might allow LRP to function more efficiently and consequently minimize the abnormal accumulation of VLDL remnants in the plasma of LDLR-deficient mice.

A reduction in the secretion of hepatic VLDL into the plasma of normal mice can be achieved by treating the animals with fibrates (5). Fibrates are a class of compounds that exert their effects through the action of PPAR{alpha}, a transcription factor belonging to a subfamily of nuclear hormone receptors. Fibrates bind to and activate PPAR{alpha}, causing this transcription factor to bind to specific response elements in the regulatory regions of target genes, resulting in the modulation of their expression (6). Fibrates induce the down-regulation of genes encoding enzymes involved in hepatic fatty acid synthesis and in the up-regulation of genes encoding enzymes of the hepatic fatty acid {beta}-oxidation pathway. In combination, these effects cause a decreased availability of fatty acids for the synthesis of triglycerides (TG) that are normally incorporated into hepatic VLDL (6). Thus, in normal animals, fibrate treatment causes fewer VLDL particles to be assembled and secreted explaining, at least in part, the well known hypotriglyceridemic effects of these compounds.

In the present study we set out to examine whether the accumulation of IDL/LDL in the plasma of LDLR-deficient mice fed a common chow diet could be reduced, as in the case of normal animals, by the administration of ciprofibrate, a potent PPAR{alpha} agonist. We report that ciprofibrate treatment caused a marked decrease in the accumulation of apoB-48-carrying IDL/LDL. At the same time, however, it caused a surprising and substantial increase in the secretion and plasma accumulation of apoB-100-carrying IDL/LDL, producing a significant rise in plasma cholesterol levels. We established that the ciprofibrate-induced changes in plasma apoB-48 and apoB-100 levels resulted from a marked inhibition of the hepatic apoB mRNA editing. We also report that after 150 days of treatment with ciprofibrate the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Wild-type female mice and homozygous female LDLR-deficient mice (6–8 weeks old), both on the C57BL/6J background, were purchased from Jackson Laboratories (Bar Harbor, ME). They were housed (5 animals/cage) in a temperature- and humidity-controlled room under 12-h cycles of light (6 AM–6 PM) and darkness (6 PM–6 AM) and were fed a normal rodent chow diet that contained 4% (w/w) fat and 0.4% (w/w) cholesterol (Teklad diet TD7022), purchased from Harlan Teklad (Madison, WI). Some animals received the same diet supplemented with 0.05% (w/w) ciprofibrate or fenofibrate purchased from Sigma. At specified time intervals, blood samples (40–100 µl/mouse) were collected from the tail of conscious animals into heparinized capillary tubes, always between 7 AM and 9 AM. When indicated, mice were deprived of food for 4 h before being sampled for blood from the tail. They were then injected through a tail vein with 0.2 ml of a 10% (v/v) solution of Triton WR-1339 in saline, and blood samples were collected 60 and 180 min later. During the entire period the animals had free access to water.

Plasma Lipid and Lipoprotein Analysis—Lipoproteins were fractionated using equal volumes of pooled plasma from various animals/group on tandem Superose 6 FPLC columns (ÄKTA, Amersham Biosciences). The columns were eluted with 200 mmol/liter sodium phosphate (pH 7.4), 50 mmol/liter NaCl, 0.03% (w/v) EDTA, and 0.02% (w/v) sodium azide at a flow rate of 0.4 ml/min. The content of TG and cholesterol in the eluted fractions, as well as in the plasma, was measured with a microplate assay technique using enzymatic assay reagent kits obtained from Sigma. For the detection of apoB-100 and apoB-48, aliquots of the FPLC-eluted fractions were diluted 1:2 (v/v) with denaturing sample buffer, heated at 70 °C for 10 min, and run on 3–8% Tris acetate gel using the NuPAGE electrophoresis system obtained from Invitrogen. The aliquots then were transferred to a nitrocellulose membrane using the wet electrophoretic transfer system from Bio-Rad. Immunoblotting was performed on a nitrocellulose membrane with rabbit polyclonal antibodies (Biodesign International, Saco, ME) against mouse apoB-48/apoB-100 as described previously (7). When indicated, scanning densitometry analysis of the apoB blots was performed on a Macintosh computer using the NIH Image program.

RNA Extraction and Analysis—Liver and small intestine mucosa RNA was extracted using TRI reagent (Sigma) and used for primer extension analysis of apoB RNA editing and quantitative reverse transcription-PCR analysis. Primer extension analysis of apoB RNA editing was performed as described previously (8). Real time quantitative PCR analysis used random hexanucleotide priming and amplification in an ABI PRISM 7000 using SYBR Green PCR master mix according to the manufacturer's instructions (Applied Biosystems). The primers were as follows: apobec-1, 5'-ACCACAACGGATCAGCGAAA-3' and 5'-TCATGATCTGGATAGTCACACCG-3' (product size, 72 bp); ACF, 5'-AGCCAGAATCCTGCAATCC-3' and 5'-AGCATACCTCTTCGCTTCATCC-3' (product size, 75 bp); and GAPDH, 5'-GGCAAATTCAACGGCACAGT-3' and 5'-AGATGGTGAATGGGCTTCCC-3' (product size, 70 bp). The data from the quantitative PCR were normalized to GAPDH levels in each sample.

Miscellaneous Assays—Cytoplasmic extracts from the supernatant fraction of a 100,000 x g ultracentrifugation spin of mouse liver homogenates (S100) were prepared as described originally with minor modifications (8). Aliquots of protein extracts were analyzed through denaturing SDS-PAGE and Western blotted with antisera to ACF, apobec-1, or 40-kDa heat shock protein (HSP-40) as detailed previously (8).

Assessment of Atherosclerotic Lesions—Mice were anesthetized with Nembutal intraperitoneally (0.65 mg/10 g of body mass), and their vasculature was perfused through the heart left ventricle for 2 min with phosphate-buffered saline containing 7 IU of heparin/ml and then for 5 min with 10% buffered formaldehyde. The entire aorta was removed and fixed overnight with buffered formaldehyde. After removing the adventicia and branched vessels, the aortas were stained with Sudan IV. For histological examination of the lesions, segments of the aortic arch were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Ciprofibrate on Plasma Lipids—Fig. 1 shows the plasma cholesterol and TG levels of wild-type and LDLR-deficient mice maintained on a chow diet alone or supplemented with 0.05% (w/w) ciprofibrate for up to 150 days. As anticipated, ciprofibrate treatment of the wild-type mice caused a significant decrease in plasma TG levels (p < 0.01) and had little effect on the low cholesterol levels compared with the untreated group. Also as anticipated the LDLR-deficient mice maintained on the chow diet alone displayed elevated plasma levels of cholesterol and TG compared with the wild-type mice. As in the wild-type mice, treatment of the LDLR-deficient animals with ciprofibrate resulted in a decrease in plasma TG levels, although a statistical significant difference (p < 0.01) was observed only at 12 days of treatment. In marked contrast, the already elevated plasma cholesterol levels in the LDLR-deficient mice increased further upon treatment with ciprofibrate and remained elevated throughout the treatment period. After 150 days on the ciprofibrate-containing diet, the cholesterol level in the LDLR-deficient animals was 473 ± 42 mg/dl compared with 280 ± 35 mg/dl in the untreated animals (mean ± S.D., n = 5) (p < 0.01). The ciprofibrate-induced changes in plasma lipids were not unique to that drug since similar increases in plasma cholesterol levels were observed in LDLR-deficient mice treated with fenofibrate, another PPAR{alpha} agonist. The results in Fig. 2 show that when 20-week-old female LDLR-deficient mice were treated with 0.05% (w/w) fenofibrate, their plasma cholesterol levels increased from 308 ± 25 mg/dl to 544 ± 27 mg/dl (mean ± S.D., n = 5) after 30 days of treatment. Fig. 2 also shows that 10 days after cessation of the treatment with fenofibrate the plasma cholesterol returned to base-line levels.



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  		FIG. 1.
Plasma cholesterol and triglycerides in mice treated with ciprofibrate. Wild-type and LDLR-deficient mice were maintained on a chow diet alone (open bars) or supplemented with 0.05% ciprofibrate (filled bars) for the indicated time periods. Results are expressed as means ± S.D. (n = 5). Statistically significant differences between groups (p < 0.01) are indicated by asterisks.

 



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  		FIG. 2.
Plasma cholesterol in mice treated with fenofibrate. Female LDLR-deficient mice (5 animals) were maintained on a chow diet alone or supplemented with 0.05% fenofibrate for 30 days. Blood samples were collected, and the treated animals were switched to a regular chow diet for another 10 days. Results are expressed as means ± S.D.

 
Plasma Lipids after Triton WR-1339 Injection—The lower triglyceride levels in the plasma of the ciprofibrate-treated LDLR-deficient mice (Fig. 1) is consistent with the well known effect that fibrates have in decreasing the secretion of VLDL TG into the circulation (6). This was confirmed in an experiment in which LDLR-deficient mice fed a chow diet with or without ciprofibrate for 30 days were injected with Triton WR-1339. The TG accumulation in their plasma was measured at 60 and 180 min after the injection (Fig. 3). It is apparent that the rate of TG influx into the circulation of the treated mice was markedly reduced. Since the animals were deprived of food for 4 h before the Triton injections and for the subsequent 3 h of the experiment, the secretion of triglycerides by the intestine was decreased, although not eliminated, and most of the TG that accumulated in the plasma of the Triton-injected mice were those carried by the VLDL secreted by the liver. We also measured the plasma cholesterol levels in the animals injected with Triton WR-1339. Before the injection, the plasma cholesterol levels in the untreated and ciprofibrate-treated mice were, respectively, 273 ± 17 and 427 ± 28 mg/dl (mean ± S.D., n = 4), and 3 h after the Triton injection the cholesterol levels increased by 84 ± 12 and 100 ± 14 mg/dl, respectively. Thus, unlike the influx of TG, the influx of cholesterol into the plasma of the ciprofibrate-treated LDLR-deficient mice was not significantly altered (p > 0.5) compared with the untreated animals.



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  		FIG. 3.
Triglyceride influx into the plasma of LDLR-deficient mice. Animals maintained on a chow diet with or without 0.05% ciprofibrate for 30 days were deprived of food for 4 h and then injected into a tail vein with 0.2 ml of a 10% solution of Triton WR-1339. Blood samples were collected at 1 and 3 h after the injection. All animals had free access to water but not food for the duration of the experiment. Results are expressed as means ± S.D. (n = 4). Open symbols, untreated; filled symbols, treated.

 
Lipoprotein Fractionation by FPLC—To examine how the fibrate-induced changes in plasma lipids were reflected on the plasma lipoprotein profile of the LDLR-deficient mice, pooled plasma of animals maintained on chow alone or chow supplemented with ciprofibrate for 30 days was fractionated by size exclusion chromatography. Fig. 4 shows the TG and cholesterol contents of the separated lipoprotein fractions. The VLDL and a considerable portion of the IDL/LDL (FPLC fractions 8–28) of the ciprofibrate-treated animals are notable for their reduced TG content compared with those fractions from the untreated mice. The IDL/LDL fraction of the treated animals is shifted to the right, indicating that the lipoproteins eluted more slowly from the FPLC column, suggesting that a portion of the IDL/LDL consisted of smaller particles. The plasma cholesterol in both the untreated and ciprofibrate-treated mice was associated predominantly with the IDL/LDL fraction. However, in the ciprofibrate-treated mice this fraction is broader and is also shifted to the right, again indicating the presence of smaller particles. No elution shift was observed in either the VLDL or the high density lipoprotein fractions. These findings were consistently observed in two other experiments (data not shown).



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  		FIG. 4.
FPLC of plasma from LDLR-deficient mice and apoB Western blot analysis. Equal volumes of plasma from female LDLR-deficient mice maintained on a chow diet alone (open symbols) (n = 5) or supplemented with 0.05% ciprofibrate (filled symbols) (n = 5) for 30 days were pooled, and 180 µl were subjected to gel filtration chromatography using Superose columns. Fractions (0.4 ml) were collected, and triglyceride and cholesterol concentrations were measured. Equal volumes of the specified FPLC fractions from the untreated and ciprofibrate-treated mice were submitted to electrophoresis, and Western blots were performed with rabbit polyclonal antibodies specific for mouse apoB-48 and apoB-100. HDL, high density lipoprotein.

 
Western Blot Analysis of FPLC Fractions—Fig. 4 also shows the distribution of apoB-100 and apoB-48 in the IDL/LDL fractions from both groups of animals as detected by Western blot analysis. It is readily apparent, from the entire IDL/LDL peak (combined FPLC fractions 20–40) as well as from selected FPLC fractions (fractions 21, 25, and 29), that the IDL/LDL from the ciprofibrate-treated mice contained considerably less apoB-48 and considerably more apoB-100 than the untreated animals. These findings were confirmed in two other separate experiments (data not shown). The presence of apoB-48 and apoB-100 could not be detected in the high density lipoprotein peaks of either group of animals, even after the eluted peaks (combined FPLC fractions 41–51) were concentrated 10-fold (data not shown).

Influx of ApoB-100 and ApoB-48 into the Plasma—The decrease in apoB-48 content in the IDL/LDL of the ciprofibrate mice could be explained by a more rapid rate of removal of apoB-48-carrying lipoproteins from the plasma or/and a decreased influx of apoB-48-carrying lipoproteins into the circulation. Likewise, the increase in apoB-100 content in the plasma of the ciprofibrate mice could be explained by a greater influx of apoB-100-carrying lipoproteins into plasma or/and a further decrease in the rate of IDL/LDL clearance. To examine the effects of ciprofibrate treatment on the influx of apoB-carrying lipoproteins into the plasma, we used a procedure described by Li et al. (9) with some modifications. Mice fed a chow diet with or without ciprofibrate for 30 days (4 animals/group) were deprived of food for 4 h and then injected with Triton WR-1339. Three h later blood samples were collected, and equal amounts of plasma from the animals in each group were pooled for the isolation of the VLDL fraction. While Li et al. (9) separated the VLDL by ultracentrifugation, we separated the plasma VLDL by FPLC. Because Triton WR-1339 inhibits LPL activity (10), the TG-rich lipoproteins secreted by the liver and intestine accumulate in the plasma, and their apoB content can be determined by Western blot analysis. Fig. 5a shows that the TG-rich lipoproteins (VLDL) that accumulated in the plasma of the ciprofibrate-treated mice eluted more slowly from the FPLC column, indicating that some lipoproteins in the peak were smaller than those secreted into the plasma of the untreated animals and presumably were the precursors of the smaller IDL/LDL particles (Fig. 4). Fig. 5, b and c, shows the Western blot and the densitometric analysis of the apoB-100 and apoB-48 in the plasma VLDL peak separated by FPLC. It is apparent that more of the liver-specific apoB-100 accumulated in the plasma of the ciprofibrate-treated animals, indicating an increased hepatic secretion of this apoprotein compared with the untreated LDLR-deficient mice. A substantial presence of apoB-48 was detected in the VLDL fractions of both groups of animals, but less was detected in the VLDL from the ciprofibrate-treated animals. However, because apoB-48 is continuously secreted by the intestine, even in animals deprived of food (11), the origin of apoB-48-carrying lipoproteins that accumulate in the plasma of Triton-injected animals cannot be established with certainty. We therefore used a different approach to determine whether a decreased influx of apoB-48-carrying hepatic VLDL could account for the decreased presence of apoB-48 in the IDL/LDL of the ciprofibrate-treated mice (Fig. 4).



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  		FIG. 5.
Influx of apoB-48 and apoB-100 into the plasma. Experimental conditions were as described in the legend for Fig. 3. Equal volumes of plasma samples from 5 animals/group were collected 3 h after Triton injection, pooled, and submitted to FPLC (a). The eluted fractions corresponding to the VLDL peaks of untreated mice (open circles) and ciprofibrate-treated mice (filled circles) were pooled, and aliquots were submitted to electrophoresis. Western blots (b) were performed with rabbit polyclonal antibodies specific for mouse apoB-48 and apoB-100, and the bands were submitted to densitometric scanning (c).

 
ApoB Gene Expression in LDLR-deficient Mice Treated with Ciprofibrate—We compared apoB mRNA editing in the liver and small intestine of untreated and ciprofibrate-treated LDLR-deficient mice. First, we examined apoB mRNA editing in the liver and intestine of LDLR-deficient mice fed a chow diet with or without ciprofibrate for 30 days. In the untreated animals apoB-48 RNA accounted for ~60 and 90%, respectively, of the hepatic and intestinal apoB mRNA population (Fig. 6, lanes 4–6). In contrast, ciprofibrate-treated mice showed a significant decrease in hepatic apoB mRNA editing (25% apoB-48; Fig. 6, lanes 1–3) but had no effect on the extent of intestinal apoB mRNA editing.



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  		FIG. 6.
Endogenous hepatic and intestinal apoB mRNA editing in control and fibrate-treated mice. RNA was extracted from the liver and small intestine of LDLR-deficient mice untreated or treated with 0.05% ciprofibrate for 30 days. Upper panels, apoB mRNA editing was determined by primer extension analysis. The primer extension products were separated on a 10% acrylamide gel containing 8 M urea and were autoradiographed. A representative of three independent experiments is shown. Lanes 1–3, fibrate-treated (F); lanes 4–6, untreated control (C). The locations of the primer (P) and of the unedited (C, apoB-100) and edited (U, apoB-48) products are indicated to the right of the gels. Lower panels, bar graph summarizing the data (mean ± S.E.; n = 5) for each group.

 
Expression of the ApoB mRNA Editing Core Components, apobec-1 and ACF—ApoB mRNA editing requires a minimal core complex composed of apobec-1, the catalytic deaminase, and ACF, the RNA-binding subunit (2). We performed real time reverse transcription-PCR to quantify these mRNAs in the livers of LDLR-deficient mice treated with or without ciprofibrate. Fig. 7 shows that there was no change in apobec-1 mRNA abundance in the livers of the LDLR-deficient treated animals compared with the untreated mice. By contrast, ciprofibrate treatment caused a significant decrease in ACF mRNA abundance. As anticipated, no changes were observed in either apobec-1 or ACF mRNA abundance in the intestine of the treated mice compared with the untreated animals. To examine apobec-1 and ACF protein expression, we also performed Western blot analysis of hepatic S100 extracts from the treated and untreated LDLR-deficient mice. Fig. 8 shows that apobec-1 protein content was comparable in both groups. In contrast, hepatic ACF protein expression was greatly decreased in the treated animals. In combination, these results clearly demonstrate that the reduced hepatic secretion of apoB-48 and the increased secretion of apoB-100 into the plasma of the ciprofibrate-treated mice were a direct consequence of an inhibition of hepatic apoB editing.



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  		FIG. 7.
Effect of fibrate treatment on apobec-1 and ACF mRNA abundance in the liver and intestine. Transcript abundance was determined by real time quantitative PCR analysis as described under "Experimental Procedures." The data (n = 5 animals/group) were normalized to GAPDH levels in each sample. The data are expressed as normalized mRNA abundance relative to control (untreated) animals. The asterisk indicates a significant difference (p < 0.001) in ACF mRNA expression in the livers of treated compared with untreated mice.

 



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  		FIG. 8.
Effect of fibrate treatment on apobec-1 and ACF protein expression. Liver S100 extracts (100 µg) obtained from the indicated groups were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with specific polyclonal antisera to apobec-1 and ACF or HSP-40 (control) and was visualized by enhanced chemiluminescence. Migration markers are indicated on the left. This is a representative of three independent experiments.

 
Atherosclerotic Lesions in LDLR-deficient Mice Treated with Ciprofibrate—Mice that lack the LDLR and express both apoB-48 and apoB-100 do not develop significant atherosclerosis when maintained on a regular chow diet (4). In contrast LDLR-deficient mice engineered to express only apoB-100 (LDLR-/-/ApoB100/100) and maintained on a similar diet develop extensive atherosclerosis (12). Prompted by our observations that ciprofibrate treatment of LDLR-deficient mice caused substantially higher plasma levels of apoB-100 than those found in the untreated mice, we examined the aorta of these animals after they had been maintained for 150 days on a chow diet supplemented with or without ciprofibrate. Fig. 9 shows the arch segment of representative aortas from two treated and one untreated mouse stained for lipids with Sudan IV. It is clear that as a result of ciprofibrate treatment these animals developed multiple lipid-laden lesions. Histological sections (Fig. 9) revealed lesions filled with foam cells and the presence of cholesterol clefts. As anticipated, atherosclerotic lesions were not observed in the untreated LDLR-deficient mice or in the treated and untreated wild-type mice (results not shown).



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  		FIG. 9.
Gross and microscopic lesions in mice treated with ciprofibrate. LDLR-deficient mice were fed a chow diet without (a) or with (b and c) 0.05% ciprofibrate for 150 days. Their dissected aortas were stained with Sudan IV. Photomicrographs of representative lesions in the aortas from the mice treated with ciprofibrate are shown in d (x20 magnification) and e (x40 magnification). The arrow indicates cholesterol clefts in the lesion.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Fibrates, through activation of PPAR{alpha}, affect the expression of multiple genes involved in the metabolism of plasma lipoproteins (13). In this report we show for the first time that ACF, the RNA-binding subunit of the core apoB RNA-editing holoenzyme, undergoes metabolic regulation in mice in response to treatment with a fibrate. As a result, the hepatic secretion of apoB-48-carrying VLDL into the plasma of LDLR-deficient mice is greatly decreased, and the secretion of apoB-100-carrying VLDL is increased. Similar effects on hepatic apoB secretion were also observed in wild-type mice treated with fibrates.2 However, unlike the LDLR-deficient mice used in the present study, an increased secretion of apoB-100-carrying VLDL in wild-type animals did not result in the accumulation of IDL/LDL in the plasma because these lipoprotein remnants are efficiently cleared from the plasma via the LDLR pathway. In mice lacking the LDLR, by contrast, the plasma clearance of apoB-100-carrying IDL/LDL is impaired because their uptake by hepatocytes depends on the LRP, which preferentially binds apoB-48-carrying lipoproteins of either intestinal or hepatic origin (14). Thus, an increased influx of apoB-100-carrying VLDL into the circulation of LDLR-deficient mice caused by fibrate treatment, as reported here, resulted in a greater accumulation of apoB-100-carrying IDL/LDL in the plasma. Other investigators (15, 16) have reported on the effects of PPAR{alpha} agonists on the in vitro secretion of apoB-100 and apoB-48 by isolated rat hepatocytes. Those studies demonstrated that compared with untreated cells, the overall secretion of apoB-100 by the PPAR{alpha} agonist-treated hepatocytes was increased, but unlike the findings reported here, the secretion of apoB-48 remained unchanged. The in vitro studies also showed that most of the apoB-100 and apoB-48 secreted was associated with lipoproteins of greater density than VLDL. The reasons for the discrepant observations between the isolated rat hepatocytes (15, 16) and those presented here are not obvious but very likely reside in the differences in experimental systems used.

In addition to a greater influx of apoB-100, a decreased rate in the removal of apoB-100-carrying IDL/LDL also may have contributed to the increased accumulation of these lipoproteins in the plasma of the ciprofibrate-treated LDLR-deficient mice. There is substantial evidence that the scavenger receptor class B, type I (SR-BI), whose main function is believed to be mediating the selective uptake of high density lipoprotein cholesterol esters, can also recognize and bind LDL (17). Wild-type and LDLR-deficient mice with liver-specific overexpression of SR-BI have been shown to have decreased levels of plasma LDL cholesterol and apoB-100, clearly suggesting that this receptor may function in the removal of the LDL particles from circulation by the liver (18–21). In the present study we found that as is the case with wild-type mice and apoE-deficient mice (7, 22), fibrate treatment of LDLR-deficient mice caused the down-regulation of SR-BI protein expression (data not shown). It is noteworthy, however, that in a study using mice with overexpression of SR-BI by adenoviral vector, Webb et al. (23) concluded that this receptor plays only a minor role in LDL metabolism in vivo. Further work is clearly necessary to determine whether SR-BI functions in the clearance of LDL from circulation and also to determine whether the decreased amounts of SR-BI in ciprofibrate-treated LDLR-deficient mice contribute to the plasma accumulation of apoB-100-carrying IDL/LDL.

Previous studies by Powell-Braxton et al. (24) using mice deficient in the LDLR and apobec-1 demonstrated that these animals, which cannot edit apoB mRNA and therefore express exclusively apoB-100, had elevated LDL cholesterol and developed extensive atherosclerosis even when maintained on a chow diet, suggesting that these animals could be used as models for human familial hypercholesterolemia. In the present study, the editing of apoB mRNA by the livers of the ciprofibrate-treated LDLR-deficient mice was only partially inhibited, and consequently, the secretion of apoB-48-carrying VLDL by the livers of these animals was greatly reduced but not completely abolished. Nevertheless, as in the study by Powell-Braxton et al. (24), this fibrate-induced inhibition of apoB mRNA editing was sufficient to cause an increase in the plasma apoB-100-carrying IDL/LDL and in the development of atherosclerotic lesions. It is possible that the ciprofibrate-induced inhibition of SR-BI expression also may have contributed to the development of atherosclerotic lesions. A decreased expression (25) or complete ablation (26) of SR-BI has been shown to result in increased severity of atherosclerotic lesions in LDLR-deficient mice. However, this phenomenon was observed in animals fed atherogenic diets (25, 26). Whether the decreased SR-BI expression in LDLR-deficient mice fed a normal chow diet, as in the present study, can promote or contribute to the development of atherosclerotic lesions remains to be determined.

It bears emphasis that in humans and other species in which hepatic apoB mRNA editing does not occur, treatment with fibrates does not promote the development of atherosclerosis. Indeed, clinical studies have provided compelling evidence that in patients at risk of developing cardiovascular disease, treatment with fibrates can retard the progression of atherosclerosis (27, 28). However, the mechanisms responsible for this outcome are presently a matter of speculation. It has generally been assumed that the beneficial effects of PPAR{alpha} agonists are due to their hypolipidemic effects. However, because PPAR{alpha} is known to be expressed in the major cell types found in atherosclerotic lesions, e.g. endothelial cells, macrophages, and smooth muscle cells (13), it has been suggested that in addition to their beneficial hypolipidemic effects, the antiatherogenic effects of PPAR{alpha} agonists may also result from the modulation in the expression of anti-inflammatory genes in those cells. In the present study we did not determine whether the expression of anti-inflammatory genes in the aortas of the LDLR-deficient mice was affected by the administered ciprofibrate. It is apparent, however, that in the presence of an enhanced hypercholesterolemia, ciprofibrate treatment did not prevent but in fact promoted the development of atherosclerotic lesions. A previous study from this laboratory (29) with another model of atherosclerosis, the apoE-deficient mouse, also showed that treatment of those animals with ciprofibrate failed to arrest the development of atherosclerosis. Duez et al. (30) reported that treatment of apoE-deficient mice with fenofibrate, although not enhancing, failed to reduce atherosclerotic lesion surface area in the aortic sinus. These authors did report a decrease in the cholesterol content in the descending aortas of the fenofibrate-treated animals, but they did not report on changes in the surface area of the lesions.

In conclusion, the present findings demonstrate that treatment of LDLR-deficient mice with fibrates results in an over-accumulation of apoB-100-carrying IDL/LDL in the plasma, causing an enhanced hypercholesterolemia and the development of atherosclerotic lesions in the aorta. As alluded to above, it is well known that hypolipidemic drugs that are effective in humans do not always have similar effects in animal models of dyslipidemia (31). The results of the present investigation, as well as our previous study with apoE-deficient mice (29) showing that treatment with fibrates aggravates the hypercholesterolemia and promotes the development of atherosclerotic lesions, are further examples of divergent effects of hypolipidemic drugs in humans and experimental animals. The fact that the fibrate-treated LDLR-deficient mice developed atherosclerotic lesions while maintained on a chow diet suggests that these animals may be useful models for atherosclerosis studies. As is often the case with human subjects, the development of atherosclerotic lesions was associated with elevated levels of apoB-100-carrying lipoprotein remnants.


   FOOTNOTES
 
* This work was supported in part by a Merit Review Grant from the Department of Veterans Affairs (to J. B.), by the Sidney and Bess Eisenberg Memorial Fund (to J. B.), by Grants HL-38180 and DK-56260 from the National Institutes of Health (to N. O. D.), and by Digestive Disease Research Center Core Grant DK-52574 from the National Institutes of Health (to N. O. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Feinberg School of Medicine, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8590; Fax: 312-503-8240; E-mail: jbb{at}northwestern.edu.

1 The abbreviations used are: apo, apolipoprotein; ACF, apobec-1 complementation factor; FPLC, fast protein liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IDL, intermediate density lipoprotein(s); LDL, low density lipoprotein(s); LDLR, low density lipoprotein receptor; LRP, low density lipoprotein receptor-like protein; PPAR, peroxisome proliferator-activated receptor; SR-BI, scavenger receptor class B, type I; TG, triglyceride(s); VLDL, very low density lipoprotein(s). Back

2 T. Fu and J. Borensztajn, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. P. Kashireddy, Dr. V. Stellmach, and Mona Cornwell for invaluable assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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