J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.M500390-JLR200 on September 26, 2005

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Journal of Lipid Research, Vol. 46, 2595-2604, December 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Loss of functional farnesoid X receptor increases atherosclerotic lesions in apolipoprotein E-deficient mice

Elyisha A. Hanniman*, Gilles Lambert{dagger}, Tanya C. McCarthy* and Christopher J. Sinal1,*

* Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
{dagger} University of Nantes Medical School, Institut National de la Santé et de la Recherche Médicale U539, Nantes, France

Published, JLR Papers in Press, September 26, 2005. DOI 10.1194/jlr.M500390-JLR200

1 To whom correspondence should be addressed. e-mail: csinal{at}dal.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The farnesoid X receptor (FXR) is a bile acid-activated transcription factor that regulates the expression of genes critical for bile acid and lipid homeostasis. This study was undertaken to investigate the pathological consequences of the loss of FXR function on the risk and severity of atherosclerosis. For this purpose, FXR-deficient (FXR–/–) mice were crossed with apolipoprotein E-deficient (ApoE–/–) mice to generate FXR–/–ApoE–/– mice. Challenging these mice with a high-fat, high-cholesterol (HF/HC) diet resulted in reduced weight gain and decreased survival compared with wild-type, FXR–/–, and ApoE–/– mice. FXR–/–ApoE–/– mice also had the highest total plasma lipids and the most atherogenic lipoprotein profile. Livers from FXR–/– and FXR–/– ApoE–/– mice exhibited marked lipid accumulation, focal necrosis (accompanied by increased levels of plasma aspartate aminotransferase), and increased inflammatory gene expression. Measurement of en face lesion area of HF/HC-challenged mice revealed that although FXR–/– mice did not develop atherosclerosis, FXR–/–ApoE–/– mice had approximately double the lesion area compared with ApoE–/– mice.

In conclusion, loss of FXR function is associated with decreased survival, increased severity of defects in lipid metabolism, and more extensive aortic plaque formation in a mouse model of atherosclerotic disease.

Abbreviations: apoC-II, apolipoprotein C-II; AST, aspartate aminotransferase; FPLC, fast-performance liquid chromatography; FXR, farnesoid X receptor; H+E, hematoxylin and eosin; HF/HC, high-fat/high-cholesterol; MAC1, macrophage antigen 1; RCT, reverse cholesterol transport; TNF-{alpha}, tumor necrosis factor-{alpha}

Supplementary key words atherosclerosis • nuclear receptor • liver


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dyslipidemia, characterized by low levels of HDL and high levels of LDL, is a known risk factor for the development of atherosclerosis. An important early event in this disease process is the deposition of lipid-filled plaques in the walls of arteries. Plaque formation is initiated when arterial wall macrophages accumulate modified LDL and initiate a chronic inflammatory state. Over time, this inflammatory state can lead to thickening of the arterial wall, fibrosis, and eventually rupture of the plaque, thrombosis, and occlusion of normal blood flow (1). The current regimen of pharmacological therapies for the treatment of cardiovascular disease remains incomplete, despite the fact that cardiovascular disease (characterized by atherosclerosis) remains the leading cause of death in Western societies (2, 3). Nuclear receptors are ligand-activated transcription factors that regulate many physiological and developmental processes, such as reproduction, metabolism, and cellular differentiation. This function is achieved by modulating the expression of target genes through binding to specific response elements in the promoter regions of these genes, leading to the recruitment of coactivators or corepressors that induce or repress the target gene, respectively (4). A number of nuclear receptors, including the liver X receptors and peroxisome proliferator-activated receptors, have important regulatory roles in lipid homeostasis (5–8). Consequently, a great deal of research has been devoted to the selective modulation of these receptors as a novel therapeutic approach to the prevention and treatment of atherosclerosis (9–11).

Excess cholesterol is transported from peripheral tissues to the liver by HDL-mediated reverse cholesterol transport (RCT). Once in the liver, cholesterol can be secreted into the bile either directly or after conversion to bile acids. The farnesoid X receptor (FXR) is a nuclear receptor that is activated by binding to bile acids and functions primarily as a bile acid sensor in the liver to induce and repress genes involved in bile acid export (bile salt export pump) and synthesis (cytochrome P450 7A1), respectively (12, 13). This conversion of cholesterol to bile acids and subsequent efflux to the bile is a quantitatively important contributor to RCT and by extension is also important for the prevention of atherosclerosis. Beyond this function, accumulating evidence strongly supports an expanded role for FXR in the regulation of systemic lipid homeostasis. For example, FXR activates the expression of the genes encoding apolipoprotein C-II (apoC-II) and the LDL receptor (14, 15) and represses the expression of the genes encoding apoA-I and apoC-III as well as hepatic lipase (16, 17). More recently, FXR has also been linked to the regulation of carbohydrate metabolism (18–20). Together, these data indicate a broad regulatory role for FXR in systemic energy metabolism.

Given that FXR activation is linked to the repression of hepatic bile acid synthesis, agonists of this receptor would be expected to impair RCT and hepatic cholesterol elimination. Consistent with this, treatment of wild-type but not FXR–/– mice with the FXR antagonist guggulsterone has been reported to decrease hepatic cholesterol (21, 22). Paradoxically, FXR–/– mice exhibit increased blood triglyceride and cholesterol levels as well as increased accumulation of lipids in the liver, despite increased secretion of both bile acids and cholesterol (23, 24). Furthermore, FXR agonists have been shown to produce a potentially beneficial reduction of blood triglyceride in rodents (14, 17, 25). This study was undertaken to examine and clarify the pathological consequences of the loss of FXR function on lipid homeostasis and atherosclerotic progression. The results of this study indicate that targeted disruption of the FXR gene leads to more severe atherosclerosis in an established murine model of this disease and provide the first direct evidence linking this receptor to the risk and severity of cardiovascular disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and diets
ApoE–/– mice were purchased from Jackson Laboratories (Bar Harbor, ME). FXR–/– mice (from our breeding colony, backcrossed 10 generations to congenic C57BL/6J mice) were crossed with ApoE–/– mice to obtain the FXR–/–ApoE–/– mice. The background of all mice used in this study was C57BL/6J. Seven- to 8-week-old male mice were placed on either chow (7% fat) (F4516; based on the AIN-93G diet) or a high-fat, high-cholesterol (HF/HC) diet containing 16% fat and 1.25% cholesterol (F4515; Bioserv, Frenchtown, NJ) for 12 weeks. Animals were weighed once per week, and food consumption was monitored throughout the study. All animals were housed at room temperature on a 12 h light/dark cycle and provided food and water ad libitum. All procedures were conducted at the Carleton Animal Care Facility in accordance with Canadian Council on Animal Care guidelines.

Plasma analyses
Blood was collected using heparinized needles after a 6 h fast and centrifuged at 6,700 g for 5 min. Plasma was stored at –20°C. Triglyceride and cholesterol levels were measured at 510 and 500 nm, respectively, using in vitro diagnostic reagents and calibrators (Thermo DMA, Arlington, TX) according to the manufacturer's instructions. Aspartate aminotransferase (AST) values were measured after dilution of the plasma 1:5 with distilled water and using an Infinity AST Reagent kit (Sigma Diagnostics, St. Louis, MO). Values were determined kinetically by the change in absorbance at 340 nm and at a temperature of 37°C. All absorbance measurements were performed using a 96-well plate reader (PowerWaveX; Bio-Tek Instruments, Inc., Winooski, VT). Blood glucose was measured using a personal glucose monitoring device (TheraSense FreeStyle; AR-Med, Ltd.).

Fast-performance liquid chromatography and Western blot analysis
Fast-performance liquid chromatography (FPLC) separation of plasma lipoproteins from pooled plasma samples (200 µl; n = 3–5) and analysis of lipid content of the subsequent fractions were performed as described previously (26). Immunoblot analyses of apoB-100, apoB-48, apoA-I, and apoA-II in the VLDL, LDL, and HDL fractions (eluting at 14, 20, and 29 ml, respectively) were performed as described previously (24).

Liver lipid measurement
Lipids were extracted from liver tissue based on published methods (27). Solvents were evaporated from the extracted lipids under nitrogen and dissolved in Triton X-100 (Sigma-Aldrich, St. Louis, MO), warmed to 37°C, and mixed with water (1:4). The samples were then analyzed for triglyceride and cholesterol content using the same reagents (1 µl of sample:100 µl of reagent) listed above for plasma lipid analysis.

Histology
After 12 weeks on the diets, the largest lobe of the liver and the top half of the heart were obtained from the mice and embedded in OCT (Sakura Finetek U.S.A., Inc., Torrance, CA) combined with sucrose (20%) and stored at –80°C. Ten micrometer cryosections were obtained lengthwise through the liver lobe and 5 µm cryosections were obtained in cross-section through the aorta at the origin in the heart. All cryosections were fixed for 1 min in formaldehyde solution, stained for 10 min with Oil Red O (stains lipids red), and counterstained for 1 min with hematoxylin (stains nuclei blue) for qualitative observation of lipid accumulation. Five micrometer thick cross-sections were obtained from paraffin-embedded liver and stained with hematoxylin and eosin (H+E). En face lipid accumulation was determined by removing the aortas from the mice from the ileal bifurcation to the origin at the heart. The heart and the aorta were fixed for a minimum of 2 days in 5% neutral buffered formalin (EM Industries, Inc., Gibbstown, NJ) and 0.5x phosphate-buffered saline. The aortas were then cut longitudinally, splayed and pinned in a dish that was flooded with Sudan IV stain (stains lipids red) for 8 min, destained in 80% ethanol for 5 min, and photographed. Quantitation of plaques from the ileal bifurcation to the origin (not including any branching vessels) was performed using freehand selection of plaques using ImageJ software (National Institutes of Health, Bethesda, MD). The additive area of all the plaques in a given aorta was calculated as a percentage of the total surface area of the aorta.

Hepatic gene expression
Total hepatic RNA was isolated using Trizol reagent according to the supplier's instructions (Invitrogen, Carlsbad, CA). Quantitative RT-PCR analysis was performed as described previously (28) with the following modifications. Total RNA (2 µg) was reverse-transcribed using Stratascript reverse transcriptase (Stratagene, La Jolla, CA) with random hexamers pd(N)6 according to the supplier's instructions. The synthesized cDNA was then amplified by quantitative PCR using a Stratagene MX3000p thermocycler in a total volume of 25 µl with Brilliant SYBR Green QPCR Master Mix. Primer sequences are as follows: murine tumor necrosis factor-{alpha} (TNF-{alpha}), 5'-CCC TCA CAC TCA GAT CAT CTT CT-3' (forward) and 5'-GCT ACG ACG TGG GCT ACA G-3' (reverse) (accession number NM_013693); murine macrophage antigen 1 (MAC1), 5'-GTG GTG CAG CTC ATC AAG AA-3' (forward) and 5'-GCC ATG ACC TTT ACC TGG AA-3' (reverse) (accession number M31039); and murine RNA polymerase II, 5'-CTG GAC CTA CCG GCA TGT TC-3' (forward) and 5'-GTC ATC CCG CTC CCA ACA C-3' (reverse) (accession number U37500). Thermal cycling conditions were identical for each primer pair and were as follows: a single cycle of 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 18 s, and elongation at 72°C for 30 s. Melting curves were generated from 60°C to 94°C at the end of the PCR protocol to ensure the amplification of a single product. The PCR products were then separated on a 2.5% agarose gel and visualized by ethidium bromide staining to ensure that a single product at the appropriate size was generated. Relative threshold cycle (CT) values were obtained by the {Delta}{Delta}CT method (29) using a threshold of 10 standard deviations above background for CT.

Statistics
All comparisons were performed within diet groups using one-way ANOVA with Tukey-Kramer postanalysis, unless stated otherwise. GraphPad Instat (Instat3 version 3.0a; GraphPad Software, Inc.) was used for all statistical analyses. Values are expressed as means ± SD. Differences between groups are considered statistically significant at P <= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FXR–/–ApoE–/– mice have a decreased survival rate and fail to gain weight on a HF/HC diet
To study the effect that deletion of FXR has on atherosclerotic disease, FXR–/– mice were crossed with ApoE–/– mice to generate FXR–/–ApoE–/– animals. Before crossing with the ApoE–/– mice (pure C57BL/6J background), the existing FXR–/– mouse model (24) was backcrossed 10 generations to a congenic C57BL/6J background. We then fed these mice standard rodent chow or the HF/HC diet containing increased levels of fat (16%, w/w) and cholesterol (1.25%, w/w) for a total of 12 weeks. Cholic acid is commonly included in atherogenic diets to increase the dietary absorption of lipids and to increase the inflammatory response. Because we have shown previously that dietary cholic acid is extremely toxic to FXR–/– mice (24), we omitted this component from our atherogenic diet. As indicators of the general health of mice throughout the 12 week feeding study, starting weights (day 1), cumulative weight gain, food consumption, and survival rates were monitored. Starting weights were similar in all experimental groups with the exception of FXR–/–ApoE–/– mice in the chow-fed group, which were of lower starting weight than ApoE–/– mice on the same diet (Table 1). FXR–/– ApoE–/– mice on a HF/HC diet failed to gain weight to a similar magnitude as wild-type, ApoE–/–, and FXR–/– mice on the same diet. FXR–/–ApoE–/– mice on the HF/HC diet gained ~2.8-fold less weight than FXR–/– and ApoE–/– mice and 3.7-fold less than wild-type mice on the same diet (Table 1). The reduced weight gain exhibited by FXR–/–ApoE–/– mice was not attributable to reduced caloric intake, because the food consumption of this group did not differ from that of wild-type or FXR–/– mice (Table 1). ApoE–/– mice fed a HF/HC diet had the highest food consumption, consuming ~1.3-fold more than the other groups on the same diet. Survival rates of the four genotypes of mice on both chow and the HF/HC diet were 100% with the exception of the FXR–/–ApoE–/– mice on the HF/HC diet, which exhibited 66.7% survival (Table 1).


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  		TABLE 1. Reduced survival and increased plasma lipids in FXR–/–ApoE–/– mice

 
FXR–/–ApoE–/– mice have increased blood levels of cholesterol, triglyceride, and AST
After 12 weeks on regular chow, blood cholesterol levels of ApoE–/– and FXR–/–ApoE–/– mice were significantly greater than those of wild-type or FXR–/– mice fed the same diet but did not differ significantly from one another. FXR–/–ApoE–/– mice challenged by a HF/HC diet had significantly greater cholesterol levels than all other genotypes on the same diet, with blood concentrations ~11-, 6-, and 2-fold greater than those of wild-type, FXR–/–, and ApoE–/– mice, respectively (Table 1). After 12 weeks on chow, triglyceride levels in both FXR–/– and ApoE–/– mice were ~2-fold greater than those of wild-type mice on the same diet. FXR–/–ApoE–/– triglyceride levels remained significantly higher than those of the other genotypes on the same diet, with blood concentrations ~5-, 2-, and 2-fold greater than in wild-type, FXR–/–, and ApoE–/– mice, respectively (Table 1).

After 12 weeks on a HF/HC diet, blood levels of triglycerides in both FXR–/– and FXR–/–ApoE–/– mice were ~3- and 2-fold greater than in wild-type and ApoE–/– mice, respectively. Plasma AST levels for chow-fed FXR–/–ApoE–/– mice were ~4-fold greater compared with those in wild-type and ApoE–/– mice fed the same diet (Table 1). FXR–/–ApoE–/– mice also had significantly greater plasma AST levels compared with chow-fed FXR–/– mice. HF/HC feeding led to a general increase of plasma AST levels in all of the genotypes studied. Similar to the chow-fed animals, the greatest plasma AST levels measured were those of FXR–/– and FXR–/–ApoE–/– mice. Blood glucose levels did not differ significantly between groups at week 12 of the feeding study (Table 1).

FXR–/–ApoE–/– mice have a more severe proatherogenic plasma lipoprotein profile
Further characterization of alterations in blood lipids caused by the deletion of FXR in the ApoE–/– mouse involved analysis of the lipoprotein profiles of these mice by FPLC (Fig. 1) . Fractions obtained at elution volumes of 14, 20, and 29 ml (corresponding to the VLDL, LDL, and HDL2/3 peaks, respectively) were further characterized by immunoblotting for apolipoprotein content (Fig. 1A, B, insets). On either chow or the HF/HC diet, FXR–/– mice had increased VLDL and LDL lipids as well as reduced HDL2/3 cholesterol, apoA-I, and apoA-II compared with controls. The HDL peak was shifted toward smaller elution volumes (26–28 ml), indicating the presence of large triglyceride-rich HDL1 particles only in the HF/HC-fed FXR–/– mice (Fig. 1A, C). In the ApoE–/– background, FXR deficiency increased the levels of VLDL apoB-48 and decreased those of HDL apoA-I and apoA-II. This was exacerbated by HF/HC feeding, with dramatically increased apoB-48 associated with VLDL and LDL as well as decreased apoA-I and apoA-II associated with HDL (Fig. 1B, inset) in FXR–/–ApoE–/–mice.



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  		Fig. 1. Fast-performance liquid chromatography (FPLC) and Western blot analysis of plasma lipoprotein and apolipoprotein content. FPLC cholesterol (A) and triglyceride (C) profiles of wild-type mice fed chow (blue) or a high-fat/high-cholesterol (HF/HC; red) diet and of farnesoid X receptor-deficient (FXR–/–) mice fed chow (yellow) or HF/HC (green). FPLC cholesterol (B) and triglyceride (D) profiles of apolipoprotein E-deficient (ApoE–/–) mice fed chow (blue) or HF/HC (red) and of ApoE–/–FXR–/– mice fed chow (yellow) or HF/HC (green). The insets show apolipoprotein Western blot analyses performed on fractions obtained from FPLC: V, VLDL; L, LDL; H, HDL. Serum (200 µl) was pooled from three to five animals for each experimental group.

 
Examination of the lipoprotein distribution patterns for cholesterol in the plasma of chow-fed FXR–/–ApoE–/– and ApoE–/– mice revealed that FXR–/–ApoE–/– mice had the highest amount of cholesterol in the VLDL fractions. HF/HC feeding of FXR–/–ApoE–/– and ApoE–/– mice further increased plasma cholesterol in the form of VLDL in both genotypes. The levels of VLDL and LDL cholesterol were highest in FXR–/–ApoE–/– mice compared with the other genotypes (Fig. 1B). In addition, FXR–/–ApoE–/– animals had sharply increased VLDL triglyceride levels compared with ApoE–/– animals on both diets. Surprisingly, plasma triglycerides, eluting mostly in the VLDL range, were maximal in FXR–/–ApoE–/– mice on both chow and the HF/HC diet (Fig. 1D).

FXR–/–ApoE–/– mice challenged with a HF/HC diet exhibit hepatic lipid accumulation, focal necrosis, and inflammation
Given the importance of the liver in lipid homeostasis and RCT, liver histology (H+E examination for necrosis) in HF/HC-fed groups in addition to levels of triglyceride and cholesterol (in both chow- and HF/HC-fed mice) were examined. H+E staining of liver from HF/HC-fed mice revealed that both FXR–/– and FXR–/–ApoE–/– mice had areas of focal necrosis, as indicated by localized accumulation of cells (most likely neutrophils and/or activated lymphocytes; Fig. 2A , arrowheads) (30). Similar indications of necrosis were not evident in the livers from HF/HC-fed wild-type and ApoE–/– mice. A qualitative approach to examine the hepatic accumulation of lipid was used by staining 10 µm sections of liver with Oil Red O (stains lipids red; Fig. 2A). Livers from FXR–/– and FXR–/– ApoE–/– mice have increased lipid accumulation on a HF/HC diet compared with wild-type and ApoE–/– mice on the same diet (seen as large red vacuoles present in liver sections).



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  		Fig. 2. Analyses of hepatic lipid accumulation, necrosis, and inflammation. A: Hematoxylin and eosin (H+E)-stained sections (5 µm) of liver from HF/HC-fed mice (100x). Insets are 400x. Arrowheads indicate the accumulation of immune cells. B: Oil Red O-stained sections (5 µm) of liver from HF/HC mice (1,000x). C: Total hepatic cholesterol. D: Triglyceride. E: Liver body weight. Real-time quantitative PCR analysis of hepatic tumor necrosis factor-{alpha} (TNF-{alpha}; F) and macrophage antigen 1 (MAC1; G) mRNA levels. All values shown are means ± SD and were normalized to RNA polymerase II mRNA levels; they are expressed as the fold difference relative to chow-fed wild-type mice. * P < 0.05 versus wild-type mice, # P < 0.05 versus FXR–/– mice, and + P < 0.05 versus ApoE–/– mice on the same diet. n = 3 for hepatic lipids, n = 4–5 for real-time quantitative PCR. Bars = 50 µm.

 
Lipids were organically extracted from liver tissue and measured using commercial colorimetric kits for both cholesterol and triglycerides. Interestingly, HF/HC feeding of FXR–/– and FXR–/–ApoE–/– mice led to 4-fold increases in hepatic cholesterol over chow-fed mice of the same genotype compared with only 2- and 1.6-fold increases in wild-type and ApoE–/– mice, respectively (Fig. 2C). After 12 weeks on a HF/HC diet, FXR–/– and FXR–/– ApoE–/– mice had increased hepatic concentrations of cholesterol, with increases of ~2-fold over both HF/HC-fed wild-type and ApoE–/– mice (Fig. 2C). HF/HC feeding increased hepatic triglycerides in FXR–/– mice (5-fold), FXR–/–ApoE–/– mice (3-fold), and wild-type mice (2-fold) compared with the respective chow-fed controls (Fig. 2D). Similarly, both FXR–/– and FXR–/–ApoE–/– mice fed the HF/HC diet had increased levels of hepatic triglycerides compared with wild-type and ApoE–/– groups on the same diet. Hepatic triglyceride concentrations in the FXR–/– mice were 2- and 3-fold greater than those of wild-type and ApoE–/– animals on the same diet, respectively (Fig. 2D). In accordance with the increased hepatic lipid accumulation, the liver weights (as a percentage of body weight) were increased for both HF/HC-fed FXR–/– and FXR–/–ApoE–/– mice (5.4 ± 0.6 and 5.5 ± 0.6, respectively) compared with wild-type (3.9 ± 0.5) and ApoE–/– (4.3 ± 0.4) (Fig. 2E) mice fed the same diet.

In addition to histological and lipid analyses of livers from these mice, expression of the genes for TNF-{alpha} and MAC1 (markers of inflammation) was examined using real-time quantitative PCR. Chow-fed FXR–/–, ApoE–/–, and FXR–/–ApoE–/– mice had ~3- to 4-fold higher levels of TNF-{alpha} expression compared with wild-type mice fed the same diet (Fig. 2F). HF/HC feeding dramatically increased TNF-{alpha} mRNA levels in both FXR–/– and ApoE–/– mice to ~10-fold over chow-fed wild-type mice. FXR–/– ApoE–/– mice exhibited a synergistic increase in TNF-{alpha} gene expression to ~40-fold over wild-type values (Fig. 2F). Similar to the TNF-{alpha} expression patterns, both FXR–/– and ApoE–/– mice fed chow had similar mRNA expression (~5-fold over wild-type values) of hepatic MAC1 (Fig. 2G). Chow-fed FXR–/–ApoE–/– mice had ~12-fold higher expression than wild-type mice on the same diet. HF/HC feeding led to increases in MAC1 mRNA levels (~15- to 17-fold over chow-fed wild-type mice) in both FXR–/– and ApoE–/– mice. Similar to TNF-{alpha} expression, MAC1 transcript levels showed a synergistic increase in HF/HC-fed FXR–/–ApoE–/– mice (~68-fold higher than in the wild type).

Deletion of FXR results in more severe atherosclerosis in ApoE–/– mice
Determination of atherosclerosis was performed using cross-sectional as well as en face representations of aortas (stained for lipid-filled plaques) from all animals on both chow and the HF/HC diet (Fig. 3) . Examination of both en face and cross-sectional aortas from the chow-fed mice revealed that neither wild-type nor FXR–/– animals had any detectable plaques on this diet (Fig. 3A, B). Both FXR–/– ApoE–/– and ApoE–/– mice fed chow had small amounts of detectable plaques in both cross-sectional and en face representations (Fig. 3A, B). The extent of atherosclerosis in aortas from these chow-fed ApoE–/–FXR–/– and ApoE–/– mice, as determined by quantitation of en face plaques, was not significantly different (Fig. 4A) . Challenging both wild-type and FXR–/– mice with a HF/HC diet did not result in detectable atherosclerotic plaques, as indicated by cross-sectional or en face aortas from these mice (Fig. 3A, B). In contrast, HF/HC feeding of ApoE–/– and FXR–/– ApoE–/– mice resulted in large increases in the extent of atherosclerosis in aortas of these mice (Fig. 3A, B). Quantitation of atherosclerosis in en face aortas from these mice revealed that the FXR–/–ApoE–/– mice had approximately double the amount of plaques compared with the ApoE–/– mice (37.2 ± 11.4% relative to 18.2 ± 3.2%) (Fig. 4B).



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  		Fig. 3. Analysis of aortic atherosclerosis in chow- and HF/HC-fed mice. A: Oil Red O-stained cross-sections (5 µm) through aortic roots, 700 µm past the appearance of the aortic valve (1,000x). Bars = 50 µm. B: En face staining of Sudan IV-stained aortas. Arrowheads and wedges indicate atherosclerotic plaques in chow- and HF/HC-fed animals, respectively.

 


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  		Fig. 4. Quantitation of aortic atherosclerosis in chow- and HF/HC-fed mice. A: Quantitation of atherosclerotic plaques in chow-fed mice (percentage total en face aorta surface area). B: Quantitation of atherosclerotic plaques in HF/HC-fed mice (percentage total en face aorta surface area). n = 5–8. P = 0.0007 compared with ApoE–/– mice on the HF/HC diet (Mann-Whitney U-test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study investigated the pathological consequences of deficient FXR function for the development of atherosclerotic disease. Challenging mice that lacked both functional FXR and apoE genes with a HF/HC diet led to a failure to gain weight to a similar extent and decreased survival compared with wild-type, FXR–/–, and ApoE–/– mice fed the same diet. Loss of functional FXR also led to further increases of total blood cholesterol and triglyceride levels as well as increased VLDL and LDL in the ApoE–/– model. HF/HC-fed FXR–/–ApoE–/– mice experienced the greatest detrimental effects on hepatic status, as shown by massive lipid accumulation, focal necrosis, and markedly increased inflammatory gene expression and plasma AST levels. Ultimately, all of these phenotypic characteristics were associated with the most severe degree of atherosclerotic lesion formation and the lowest survivability in the HF/HC-fed FXR–/–ApoE–/– mice compared with the other genotypes studied.

Our previous work demonstrated decreased hepatic expression of the scavenger receptor class B type I in FXR–/– mice. This was associated with decreased hepatic uptake of HDL cholesterol as well as increased synthesis of highly atherogenic apoB-containing lipoproteins (23). Additionally, FXR–/– mice exhibit hyperabsorption of cholesterol (23) and triglycerides (unpublished results) from the intestine. All of these factors are believed to contribute to the proatherogenic plasma total lipid and lipoprotein profile exhibited by FXR–/– mice. ApoE is required for hepatic LDL receptor-mediated clearance of remnants of VLDL in the liver (31), and consistent with previous studies (32, 33), we observed an almost complete absence of HDL in the plasma of ApoE–/– mice. Thus, the extremely atherogenic plasma lipid and lipoprotein profile exhibited by FXR–/–ApoE–/– mice, particularly when challenged with a HF/HC diet, reflects the striking genetic interaction between the loss of function of the FXR and apoE alleles.

Histological analysis of livers from HF/HC-fed mice revealed that both FXR–/– and FXR–/–ApoE–/– mice had areas of focal necrosis that were absent from the livers of ApoE–/– and wild-type mice fed the same diet. Consistent with this observation, HF/HC-fed FXR–/– and FXR–/– ApoE–/– mice also exhibited the highest levels of plasma AST, which may have resulted from the release of this enzyme from necrotic hepatocytes. Hepatic lipid accumulation is a hallmark feature of the pathogenesis of necrotic liver diseases such as nonalcoholic steatohepatitis (34). Thus, given the massive hepatic accumulation of lipid in both FXR–/– and FXR–/–ApoE–/– mice, it is not surprising that chronic HF/HC feeding caused damage to the livers of these animals. Furthermore, FXR–/– mice present with a cholestatic phenotype characterized by increased plasma bile acids (24). This may have also contributed to the hepatic necrosis seen in FXR–/– and FXR–/–ApoE–/– mice when fed a HF/HC diet. Indicators of an altered inflammatory state in the liver included changes in expression of the immune receptor MAC1 and the inflammatory cytokine TNF-{alpha}. MAC1, also known as CD11b/CD18 and complement receptor 3, is predominantly expressed on macrophages, monocytes, neutrophils, and natural killer cells and is involved in many functions, including activation and migration of these cells (35). TNF-{alpha} is a proinflammatory cytokine released by tissue macrophages and T-cells at a site of injury and essentially acts to activate and attract other immune cells (36). TNF-{alpha} has also been shown to induce the upregulation of MAC1 in neutrophils (37). Therefore, the dramatic increase of MAC1 and TNF-{alpha} mRNA levels in the livers of HF/HC-fed FXR–/–ApoE–/– mice most likely represented the combined effects of inflammation-induced expression of these genes in hepatocytes and infiltrating cells of the immune system such as macrophages and neutrophils. In addition, increased TNF-{alpha} levels in the FXR–/–ApoE–/– mice may have added to the induction of MAC1 expression in these immune cells. Combined with the failure to gain as much body weight and reduced survival compared with the other genotypes, these data suggest that hepatic toxicity and severe dysfunction occurred in the HF/HC-challenged FXR–/–ApoE–/– mice.

Although loss of FXR function alone was not sufficient to cause atherosclerosis when mice were challenged with a HF/HC diet, the combination of FXR deficiency with that of apoE resulted in a dramatic worsening of the disease. Unlike humans, mice transport the majority of plasma lipids in HDL particles, and this is thought to contribute to the inherent resistance of this species to atherosclerotic disease. Compared with wild-type animals, FXR–/– mice exhibit a proatherogenic plasma lipoprotein profile characterized by the presence of increased amounts of highly atherogenic VLDL and LDL particles in the plasma. Therefore, it was somewhat surprising that no atherosclerosis was detected in the FXR–/– mice fed the HF/HC diet. ApoE is synthesized in large quantities by the liver, is a constituent of all lipoproteins except LDL, and functions as a ligand for receptors that clear chylomicrons and VLDL remnants from the blood (32). ApoE is also synthesized by monocytes and macrophages and is thought to promote cholesterol efflux and modulate inflammatory responses in atherosclerotic vessels (33). Thus, the increased susceptibility of ApoE–/– mice to spontaneous and diet-induced atherosclerotic lesions results from a loss of critical functions in at least two sites, the liver and macrophage. In contrast, although FXR has well-defined regulatory roles in hepatic function, the expression of this receptor in macrophages is undetectable (unpublished results) (38). Thus, despite the severe proatherogenic plasma lipid profile of FXR–/– mice, it is likely that macrophages in the vessel walls of these mice remain resistant to lipid accumulation and plaque formation. Only when the loss of functional FXR was combined with the loss of apoE was a genetic interaction resulting in more severe dyslipidemia, impaired hepatic function, and atherosclerosis revealed.

Recently, treatment with the selective FXR agonist GW4064 was shown to prevent the development of gallstones in a mouse model of the disease (39). Importantly, this study demonstrated that modulation of FXR could be used to treat a specific disease process and more generally illustrated the therapeutic potential of targeting this receptor. At present, however, the benefits of manipulating FXR function as a novel therapeutic approach for atherosclerosis are not clear. Presumably, activation of FXR in vivo would lead to decreased conversion of cholesterol to bile acids, an effect detrimental to the treatment of coronary artery disease and hyperlipidemia. However, data generated in our study as well as previous work with FXR–/– mice have demonstrated that the role of FXR in lipid homeostasis is more complicated than previously thought. For instance, FXR–/– mice exhibit increased intestinal absorption of cholesterol and triglycerides, decreased hepatic uptake of HDL, and increased synthesis of apoB-containing lipoproteins (23). All of these characteristics associated with the loss of FXR function suggest that antagonism of this receptor would lead to an undesirable disruption of systemic lipid homeostasis that ultimately may be detrimental to the treatment of atherosclerosis. In addition, it is not known whether modulation of hepatic FXR function can affect the inflammatory state at the level of the vessel wall in addition to that seen in the liver in this study. Studies of the lipid-lowering mechanism of an herbal product, guggulsterone, revealed antagonism of FXR and a decrease of hepatic cholesterol accumulation (21, 22) as well as hypolipidemic effects and increased hepatic LDL uptake (40). These findings lead to the question of why antagonism of FXR by guggulsterone is lipid-lowering but targeted disruption of the FXR gene leads to hyperlipidemia and increased atherosclerosis when combined with loss of apoE function. One answer to this controversy may be provided by recent work demonstrating that guggulsterone antagonizes a number of nuclear receptors (including glucocorticoid, mineralocorticoid, and androgen receptors) with up to 100-fold greater potency than that exhibited for FXR (41). Furthermore, guggulsterone is also a potent activator of a number of other nuclear receptors, including the estrogen, progesterone, and pregnane X receptors (42). Therefore, the in vivo effects of guggulsterone are likely to be mediated by several mechanisms other than antagonism of FXR.

In conclusion, this study demonstrates that loss of FXR function causes increased atherosclerosis in the ApoE–/– mouse model of this disease. Other outcomes precipitated by the loss of FXR in this model included decreased weight gain and survival, increased hepatic and plasma lipids, increased hepatic inflammation, and a more severe plasma lipoprotein profile. This study is the first to demonstrate a pathogenic role for FXR in atherosclerosis using an in vivo model of the disease. In contrast to other nuclear receptors with roles in macrophage lipid homeostasis (e.g., liver X receptors, peroxisome proliferator-activated receptors), the worsening of atherosclerosis caused by targeted deletion of FXR appears to be a consequence of the loss of function of this receptor in liver and gut only. Our data demonstrate the pathological consequences of a lack of FXR function and the potential for genetic interactions of deleterious mutations of the gene for this nuclear receptor with other gene mutations known to increase the risk for cardiovascular disease.


   ACKNOWLEDGMENTS
 
The authors thank Cécile Boyer and Gordon Nash for excellent technical assistance. Funding for this study was provided by the Canadian Institutes of Health Research. E.A.H. is the recipient of a studentship from the Nova Scotia Health Research Foundation.

Manuscript received August 29, 2005 and in revised form September 15, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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