Decreased Atherosclerosis in Heterozygous Low Density Lipoprotein Receptor-deficient Mice Expressing the Scavenger Receptor BI Transgene*

Takeshi Arai, Nan Wang, Mikhail Bezouevski, Carrie Welch, and Alan R. TallDagger

From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032

    ABSTRACT
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Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    EXPERIMENTAL PROCEDURES

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.

    RESULTS

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.

                              
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Table I
Plasma lipid concentration of SR-BI Tg mice in LDLRKO background
The values are expressed as mg/dl for total cholesterol, triglyceride, and HDL cholesterol (HDLC), and µg/ml for apoB, respectively. Non-HDL cholesterol was calculated by subtracting HDL cholesterol from TC. The data are shown as mean ± S.E.; n = 4-6 female mice except SR-BI Tg with LDLR0 background on a high fat/cholesterol/bile salt diet (n = 2 in each group). The footnotes give statistical significance between the control and SR-BI Tg on each diet as determined by two-tailed Student's t test for unpaired data.

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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Fig. 1.   FPLC cholesterol profiles on different diets. Plasma was taken after feeding each diet for 4 weeks. FPLC was performed on 200 µl of pooled plasma samples obtained from 4-6 mice in each group except for LDLR0 and LDLR0/SR-BI Tg on a high fat/cholesterol/bile salt diet where n = 2. The cholesterol content of each fraction was measured by enzymatic assay and calculated to indicate cholesterol concentration in plasma (mg/dl). bullet , LDLR1; open circle , LDLR1/SR-BI Tg.

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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Fig. 2.   SDS-PAGE of isolated lipoproteins of SR-BI Tg mice in LDLRKO background on different diets. The pooled plasmas of female mice (collected after 4-5 h fasting, n = 4-6 for group) on chow, high fat/cholesterol, and high fat/cholesterol/bile salt diets were used. Lipoproteins were isolated by sequential density centrifugation (Beckman, OptimaTM TL Ultracentrifuge, rotor; TLA 100.4), 1.006-1.019 for VLDL/intermediate density lipoproteins, 1.019-1.055 for LDL, and 1.055-1.210 for HDL from 200 µl of pooled plasma. The samples corresponding to 15-20 µl of plasma (A-E and HDL fraction in F) and 6 µl of plasma (VLDL/intermediate density lipoproteins and LDL fractions in F) were loaded onto reducing 4-20% SDS-PAGE gels and stained with Coomassie Brilliant Blue. Similar results were obtained in two different experiments.

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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Fig. 3.   Extent of atherosclerosis in the proximal aorta of SR-BI Tg mice in LDL receptor-deficient backgrounds. A, a high fat/cholesterol/bile salt diet was administrated to the LDLR1 and LDLR1/SR-BI Tg mice for approximately 12 weeks. Then they were sacrificed, and the heart and proximal aorta were removed. Sequential sections 10-µm-thick were made with cryostat and stained with Oil Red O. The mean area of red color lipid staining per section per animal from five sections was determined for each individual animal. The mean atherosclerosis areas are: female, 74,120 ± 18,648 µm2 for LDLR1 versus 12,667 ± 3,471 µm2 for LDLR1/SR-BI Tg, p < 0.02; male, 25,747 ± 1,616 µm2 versus 5,448 ± 2,098 µm2, p < 0.001, respectively (mean ± S.E.). B, a high fat/cholesterol/bile salt diet was administrated to LDLR0 and LDLR0/SR-BI Tg mice for 6 weeks. C, A high fat/cholesterol diet was administrated to LDLR0 and LDLR0/SR-BI Tg mice for 12 weeks. The mean atherosclerosis areas are: female, 112,285 ± 8 20,399 µm2 for LDLR0 versus 169,441 ± 31,359 µm2 for LDLR0/SR-BI Tg, not significant; male, 43,189 ± 13,707 µm2 versus 78,532 ± 16,448 µm2, not significant, respectively.

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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Fig. 4.   Correlations between VLDL plus LDL cholesterol and atherosclerosis area (A) and HDL cholesterol and atherosclerosis area (B). Individual values shown are SR-BI Tg mice in LDLRKO (i.e. LDLR1 or LDLR0) background and control LDLRKO (LDLR1 or LDLR0). The mice were fed a high fat/cholesterol or high fat/cholesterol/bile salt diet as described in the legend to Fig. 3. bullet , LDLRKO female; open circle , LDLRKO/SR-BI Tg female; black-square, LDLRKO male; , LDLRKO/SR-BI Tg male.


    DISCUSSION

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-alpha 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-alpha -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-alpha 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-alpha 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.

    ACKNOWLEDGEMENT

We are thankful for the helpful advice of Humaira Serajuddin.

    FOOTNOTES

* 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.

Dagger 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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Abstract
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