From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032
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ABSTRACT |
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Scavenger receptor type B class I (SR-BI),
initially identified as a receptor that recognizes low density
lipoprotein (LDL), was recently shown to mediate the selective uptake
of high density lipoprotein (HDL) cholesteryl esters in liver and
steroidogenic tissues. To evaluate effects on atherosclerosis,
transgenic mice with liver-specific overexpression of SR-BI (SR-BI Tg
mice) have been crossed onto LDL receptor-deficient backgrounds. To
induce atherosclerosis in a setting of moderate hypercholesterolemia, heterozygous LDL receptor-deficient mice (LDLR1) were fed a high fat/cholesterol/bile salt diet, and homozygous LDL receptor knock-outs (LDLR0) were fed a high fat/cholesterol diet. LDLR1/SR-BI Tg mice showed decreases in VLDL, LDL, and HDL cholesterol and a significant 80% decrease in mean lesion area in the aortic root compared with LDLR1 mice (female LDLR1 74, 120 µm2
versus LDLR1/SR-BI Tg 12, 667 µm2; male 25, 747 µm2 versus 5, 448 µm2,
respectively). LDLR0/SR-BI Tg mice showed decreased LDL and HDL
cholesterol but increased VLDL cholesterol and no significant difference in extent of atherosclerosis compared with LDLR0 mice. Combined data analysis showed a strong correlation between
atherosclerotic lesion area and the VLDL+LDL cholesterol level but no
correlation with HDL level. These studies demonstrate a strong
anti-atherogenic potential of hepatic SR-BI overexpression. In mice
with marked overexpression of SR-BI, the protective effect appears to
be primarily related to the lowering of VLDL and LDL cholesterol levels.
Scavenger receptor type B class I
(SR-BI),1 a member of the
CD36 gene family, was recently shown to bind HDL in a specific fashion
and to mediate the selective uptake of HDL cholesteryl esters (CE) in
cells and tissues (i.e. the uptake of CE from HDL without
degradation of HDL protein (1, 2)). SR-BI is highly expressed in liver
and steroidogenic tissues, the principal sites of selective uptake
in vivo (3-5). Mice with decreased expression of SR-BI as a
result of targeted gene mutation have increased HDL cholesterol levels
and decreased selective uptake of HDL CE in the liver (6, 7).
Overexpression of SR-BI results in decreased HDL cholesterol, increased
selective uptake of HDL CE in the liver, and an increase in biliary
cholesterol content, suggesting an enhancement of reverse cholesterol
transport (8, 9). The enhancement of reverse cholesterol transport by
SR-BI may be related both to increased selective uptake of CE in the liver, as well as increased flux of free cholesterol between HDL and
cell membranes (10).
SR-BI was originally described as a receptor recognizing both native
and acetylated LDL (11). Mice with liver-specific overexpression of
SR-BI were found to have decreases in VLDL and LDL cholesterol and
apoB, as well as reduced HDL levels, and to be resistant to increases
in apoB in response to high fat, high cholesterol diets (9). The SR-BI
Tg mice used in this study were derived from C57BL/6J × CBA/J
(9). Liver-specific overexpression of SR-BI was confirmed by Northern
analysis (9). The hepatic membrane SR-BI protein levels were shown to
be about 12-fold higher compared with control littermates (9). The
purpose of the present study was to evaluate the impact of hepatic
overexpression of SR-BI on the development of atherosclerosis. LDL
receptor knock-out mice were chosen for study because they represent an
atherosclerosis-susceptible model of the common human genetic disorder,
familial hypercholesterolemia (12). Accordingly, the SR-BI transgene
was bred onto the LDL receptor-deficient background (13), and the mice
were challenged with high fat, high cholesterol diets. To evaluate
lesion development in the context of a moderate level of
hypercholesterolemia, studies were carried out in LDLR1 mice fed a high
fat/cholesterol/bile salt diet and in LDLR0 mice fed a high
fat/cholesterol diet.
Animals--
SR-BI Tg mice were generated as described
previously (9, 10, 14). Female C57BL/6J and LDLR0 in the C57BL/6J
background were purchased from Jackson Laboratory. The founder of SR-BI
Tg (C57BL/6J × CBA/J) was backcrossed to C57BL/6J mice to N2
generation. To generate LDLR1/SR-BI Tg and LDLR0/SR-BI Tg, SR-BI Tg
(N2) was backcrossed two times to female LDLR0 mice in the C57BL/6J
background. Studies in this paper were performed using the mice with
93.75% C57BL/6J background (N4). We used the same generation
littermates as controls.
Mice were fed either chow (PMI Nutrition International, laboratory
rodent diet number 5001) or a high fat/cholesterol diet containing 20%
hydrogenated coconut oil and 0.15% cholesterol (Research Diets, Inc.
NJ) or a high fat/cholesterol/bile salt diet containing 1.25%
cholesterol, 7.5% cocoa butter, 7.5% casein, and 0.5% sodium cholate
(Research Diets, Inc., town, NJ) for 4 weeks prior to lipid analysis
and for 12 weeks for atherosclerosis studies.
Plasma Lipoprotein Analysis--
Food was removed from the cages
in the morning and blood was drawn after 4-5 h fasting. Total plasma
cholesterol and triglycerides were determined using commercial
enzymatic assays (4). Plasma HDL cholesterol was determined after
dextran sulfate-Mg2+ precipitation of apoB-containing
lipoproteins (15). Determination of plasma apoB levels was carried out
using an enzyme-linked immunosorbent assay with an affinity purified
polyclonal antibody against murine apoB (9). Non-HDL cholesterol (VLDL + LDL) was calculated by subtracting HDL cholesterol from plasma
cholesterol. Plasma lipoproteins were analyzed by fast protein liquid
chromatography (FPLC) system consisting of two Superose 6 columns
connected in series (Amersham Pharmacia Biotech) (16).
Aortic Root Assay--
LDLR1 and LDLR1/SR-BI Tg mice were fed a
high fat/cholesterol/bile salt diet for 12 weeks. LDLR0 and LDLR0/SR-BI
Tg mice were fed a high fat/cholesterol diet for 12 weeks or a high
fat/cholesterol/bile salt diet for 6 weeks. Then the animals were
sacrificed, and their hearts and aortas were collected. The hearts were
stored in 10% formalin at 4 °C before sectioning. Sequential
sections 10-µm-thick were taken using a cryostat (17). The sections
were stained in Oil Red O. The mean area of lipid staining per section
per animal from five sections was determined for each individual animal.
Statistical Analysis--
Statistical analysis was performed by
two-tailed Student's t test for unpaired data.
Plasma lipids and lipoproteins were characterized in LDLR1/SR-BI
transgenic mice and in LDLR0/SR-BI Tg mice, on chow, high fat/cholesterol, or high fat/cholesterol/bile salt diets (Table I). Compared with LDLR1 controls,
LDLR1/SR-BI Tg mice showed significant decreases in total cholesterol
(TC), HDL cholesterol, and apoB on all three diets but no difference in
triglyceride level. Similarly, compared with LDLR0 mice, LDLR0/SR-BI Tg
mice showed decreases in TC on chow and high fat/cholesterol/bile salt diets; however, there was no difference of TC on the high
fat/cholesterol diet. On the three diets, apoB levels tended to
decrease in LDLR0/SR-BI Tg compared with LDLR0 mice, but none of these
changes were significant. In LDLR0/SR-B1 Tg mice HDL cholesterol was
dramatically decreased on all diets. Plasma triglyceride levels were
increased in LDLR0/SR-BI Tg mice on chow and high fat/cholesterol diets
but not on the high fat/cholesterol/bile salt diet.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS
Plasma lipid concentration of SR-BI Tg mice in LDLRKO background
To further characterize the changes in plasma lipoprotein levels, FPLC was carried out on pooled plasma samples. Fig. 1A shows the profiles obtained in LDLR1 mice, with and without the SR-BI transgene, on the three different diets. HDL cholesterol levels were markedly reduced on all three diets. However, the response of VLDL and LDL cholesterol was diet-dependent. On the chow diet, LDL cholesterol was reduced in LDLR1/SR-B1 Tg mice. On the high fat/cholesterol diet, VLDL cholesterol was increased, whereas LDL cholesterol was unchanged. On the high fat/cholesterol/bile salt diet, both VLDL and LDL cholesterol were decreased in LDLR1/SR-B1 Tg mice compared with LDLR1 mice.
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Generally parallel but more exaggerated VLDL and LDL responses were obtained when the SR-B1 transgene was expressed in the LDLR0 background (Fig. 1B). On chow LDL cholesterol was reduced in LDLR0/SR-B1 Tg mice compared with LDLR0 mice, and there appeared also to be a small increase in VLDL cholesterol. On the high fat/cholesterol diet, VLDL was increased, but LDL cholesterol was decreased in LDLR0/SR-B1 Tg mice compared with control. On the high fat/cholesterol/bile salt diet, both VLDL and LDL cholesterol were lower in LDLR0/SR-B1 Tg mice compared with LDLR0 mice. In summary, SR-BI expression lowered HDL and LDL cholesterol in LDLR1 and LDLR0 backgrounds on all diets (with the exception of the high fat/cholesterol diet in LDLR1 animals where LDL was unchanged). In contrast, VLDL cholesterol was increased on chow or high fat/cholesterol diets but decreased on the high fat/cholesterol/bile salt diet.
To evaluate changes in the plasma apolipoproteins, SDS-PAGE analysis was performed on centrifugally isolated plasma lipoproteins, prepared from pooled plasma (Fig. 2). Expression of the SR-BI transgene resulted in a marked decrease in apoA-I on both LDL receptor-deficient backgrounds, in response to all three diets. The changes in apoB in LDL and VLDL tended to parallel the effects on LDL and VLDL cholesterol levels. Thus, in LDLR1/SR-B1 Tg mice, apoB was decreased in LDL on chow or high fat/cholesterol/bile salt diets; in the former case apoB100 was reduced, whereas in the latter case, apoB48 was reduced (Fig. 2, A and C). In the LDLR0 background, LDL apoB was decreased on chow and unchanged on high fat/cholesterol and high fat/cholesterol/bile salt diets as a result of SR-BI expression (Fig. 2, D-F). In contrast, VLDL apoB was increased on chow and high fat/cholesterol diet but decreased on the high fat/cholesterol/bile salt diet. In addition, VLDL and HDL apoE were decreased on the high fat/cholesterol/bile salt diet on the SR-BI Tg background (Fig. 2, C and F).
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The principal goal of the present study was to evaluate the effects of SR-B1 overexpression on atherogenesis. Based on our prior experience (18), we decided to evaluate the extent of atherosclerosis in mice with moderate hypercholesterolemia. Thus, we chose LDLR1 mice fed the high fat/cholesterol/bile salt diet and LDLR0 mice fed the high fat/cholesterol diet for 12 weeks; we also studied a small number of LDLR0 animals on the high fat/cholesterol/bile salt diet. On a high fat/cholesterol/bile salt diet, LDLR1/SR-BI Tg showed an 80% decrease in mean atherosclerotic lesion area in the aortic root compared with LDLR1 mice (Fig. 3A). On the other hand, LDLR0/SR-BI Tg showed no significant difference in extent of atherosclerosis compared with LDLR0 mice after 3 months on the high fat/cholesterol diet (Fig. 3C). LDLR0/SR-BI Tg were also challenged with a high fat/cholesterol/bile salt diet for 6 weeks. The atherosclerotic lesion area was reduced about 60% (Fig. 3B). These results show that in LDL receptor-deficient mice the SR-BI transgene decreased the area of atherosclerosis on a high/fat/cholesterol/bile salt diet but had no effect on a high fat/cholesterol diet.
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Overall analysis of the data (combined for LDLR1 and LDLR0 mice) showed a strong correlation between atherosclerotic lesion area and VLDL + LDL cholesterol levels for both female and male mice (Fig. 4A). As reported previously (19), female mice tended to have more severe atherosclerosis than males. In contrast, there was no relationship between the extent of atherosclerosis and HDL cholesterol levels (Fig. 4B).
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DISCUSSION |
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These studies show that SR-BI overexpression results in a marked decrease in atherosclerosis in LDL receptor-deficient mice fed the high fat/cholesterol/bile salt diet (apparently in both LDLR1 and LDLR0 backgrounds (Fig. 3, A and B)), and no change in atherosclerosis in LDLR0 mice fed the high fat/cholesterol diet (Fig. 3C). These diet/genotype combinations were chosen because they induce moderate elevations in total plasma cholesterol levels (i.e. 200-400 mg/dl) and a lipoprotein profile not dissimilar to that present in humans. The atherosclerosis results were highly correlated with effects of SR-BI overexpression on levels of VLDL plus LDL cholesterol (Fig. 4A), which were modified by the diet. Although SR-BI overexpression dramatically lowered HDL levels irrespective of diet and LDL receptor genotype, this did not appear to be a major determinant of atherosclerosis (Fig. 4B). Because VLDL+LDL cholesterol was decreased on the low fat chow diet in both LDLR1 and LDLR0 mice expressing the SR-BI transgene, these studies suggest that the therapeutic overexpression of hepatic SR-BI could be anti-atherogenic on a fat-restricted diet.
A major effect of SR-BI overexpression was the lowering of LDL cholesterol and apoB levels, including both apoB100 and apoB48 (Fig. 2). This was seen in LDLR2 (9) and LDLR1 backgrounds on chow and high fat/cholesterol/bile salt diets and in LDLR0 background on chow diet. However, effects on VLDL cholesterol and apoB were more variable. VLDL cholesterol was slightly increased on the chow diet, markedly increased on the high fat/cholesterol diet, and decreased on the high fat/cholesterol/bile salt diet. The mechanisms underlying these complex effects are uncertain. Although SR-BI has been shown to bind native LDL (11), it is not known if this leads to LDL uptake and degradation. It is tempting to suggest that the LDL lowering effects of SR-BI overexpression is related to increased removal from the circulation either of LDL itself or its precursors, i.e. VLDL remnants. A possible role of SR-BI in mediating selective uptake of CE from VLDL remnants or LDL could also potentially be involved in lowering the cholesterol content of these fractions. In contrast, the increase in VLDL cholesterol, apoB, and triglycerides that occurred on the high fat/cholesterol diet may have reflected increased VLDL secretion, perhaps due to increased hepatic uptake of fatty acids (e.g. in esterified form) by SR-BI; this effect may be exaggerated by the high fat content of the diet. The failure to observe this response on the high fat/cholesterol/bile salt diet is likely related to the bile salt component.
Although the use of the high fat/cholesterol/bile salt diet has been
criticized for its possible inflammatory effects (20), it has been
extensively used as an experimental tool to evaluate atherosclerosis in
the resistant mouse model (19, 21-23) and to evaluate lipoprotein and
atherosclerosis changes specifically in the context of decreased levels
of hepatic 7- hydroxylase activity (24). It is interesting to note
that there was a continuous correlation of atherosclerotic lesion area
and plasma cholesterol levels irrespective of the diet employed,
suggesting that effects of the bile salt component on atherosclerosis
are primarily mediated by changes in the plasma lipoproteins (Fig.
4A). The bile salt in the diet results in down-regulation of
the rate-limiting step (7-
-hydroxylase) in bile salt biosynthesis.
The increase in VLDL and LDL levels that occurs in LDLR0 mice on this
diet has been shown to be due to increased VLDL secretion and LDL
production (13) and to be ameliorated by overexpression of 7-
hydroxylase (25). It is interesting that SR-BI overexpression resulted
in the appearance of SR-BI in the bile canaliculus (8). We speculate that SR-BI plays a role analogous to 7-
hydroxylase overexpression, leading to an increased mobilization of hepatic cholesterol into bile
and compensating for the decreased activity of this enzyme in response
to the bile salt containing diet.
The finding that the outcome of atherosclerosis studies in SR-BI Tg mice is predominantly influenced by effects on VLDL and LDL cholesterol levels, although unexpected, is consistent with earlier mouse atherosclerosis studies. Thus, Young and co-workers (26) found a high correlation between extent of atherosclerosis and plasma total or non-HDL cholesterol levels in mice with different induced mutations of apoB. In contrast to these findings, reduction of HDL levels by apoA-I knock-out or cholesteryl ester transfer protein overexpression, while modifying the extent of lesions in some models, does not have a profound impact on atherosclerosis (27-29). Perhaps the lack of predominance of HDL effects in SR-BI Tg mice is related to opposing actions, i.e. the fractional clearance of HDL cholesterol by the liver is increased while HDL levels and pool size are decreased (9). This is reminiscent of the effects of cholesteryl ester transfer protein expression, which also lowers HDL while stimulating the clearance of HDL CE by the liver (30). Our transgenic mice have marked 10-40-fold overexpression of SR-BI in the liver (9). It is possible that moderate overexpression would result in a different spectrum of lipoprotein changes, and this merits further investigation.
The present results showing decreased atherosclerosis suggest that
hepatic overexpression of SR-BI or its human homolog could have
therapeutic benefits. The lowering of the atherogenic lipoprotein fraction, i.e. VLDL+LDL cholesterol and apoB levels was seen
on chow in both LDLR1 and LDLR0 mice. Thus, this therapy could work independent of LDL receptor activity and could be synergistic with
statins. In the context of a low fat diet, hepatic SR-BI overexpression
could potentially provide a method for lowering LDL cholesterol in
patients with familial hypercholesterolemia.
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ACKNOWLEDGEMENT |
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We are thankful for the helpful advice of Humaira Serajuddin.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL54591, HL58033, and HL21006 and by Lilly Research Labs and Millenium.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Molecular
Medicine, Dept. of Medicine, Columbia University, New York, NY 10032. Tel.: 212-305-4899; Fax: 212-305-5052.
The abbreviations used are: SR-BI, scavenger receptor type B class I; TC, total cholesterol; CE, cholesteryl ester; apo, apolipoprotein; LDL, low density lipoprotein(s); VLDL, very LDL; HDL, high density lipoprotein; Tg, transgenic (mice); LDLRKO, LDL receptor knockout mice; FPLC, fast protein chromatography; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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