This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kozarsky, K. F. Articles by Rader, D. J. Search for Related Content PubMed PubMed Citation Articles by Kozarsky, K. F. Articles by Rader, D. J. Related Collections Gene therapy

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:721.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Gene Transfer and Hepatic Overexpression of the HDL Receptor SR-BI Reduces Atherosclerosis in the Cholesterol-Fed LDL Receptor–Deficient Mouse

Karen F. Kozarsky; Mary H. Donahee; Jane M. Glick; Monty Krieger; Daniel J. Rader

From the Department of Molecular and Cellular Engineering (K.F.K., M.H.D., J.M.G.), Department of Medicine (D.J.R.), and Institute for Human Gene Therapy (K.F.K., M.H.D., J.M.G., D.J.R.), University of Pennsylvania Health System, Philadelphia, Pa, and the Department of Biology, Massachusetts Institute of Technology, Cambridge (M.K.). K.F. Kozarsky and M.H. Donahee are now at the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.

Correspondence to Karen Kozarsky, PhD, SmithKline Beecham, 709 Swedeland Rd, Mail Stop UW2510, King of Prussia, PA 19406. E-mail karen_f_kozarsky{at}sbphrd.com

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—HDL cholesterol levels in humans are inversely correlated with the risk of atherosclerosis. The class B scavenger receptor type I (SR-BI) is the first molecularly well-defined HDL receptor, and hepatic overexpression of SR-BI in normal mice has been shown to result in decreased plasma HDL cholesterol levels. To determine whether SR-BI overexpression is proatherogenic or is protective against atherosclerosis, LDL receptor–deficient mice were placed on a high-fat/high-cholesterol diet for 2 or 12 weeks to induce atherosclerotic lesions of different stages and then were injected with a recombinant adenovirus encoding murine SR-BI. Transient hepatic overexpression of SR-BI in mice with both early and advanced lesions significantly decreased atherosclerosis. SR-BI expression was associated with markedly decreased HDL cholesterol and either unchanged or only modestly reduced non-HDL cholesterol levels; in all experiments, the mean HDL cholesterol levels were significantly correlated with atherosclerotic lesion size. These data suggest that interventions that promote HDL cholesterol transport and lower plasma HDL cholesterol levels can suppress atherosclerosis, even when initiated after significant lesion development. Thus, stimulation of hepatic SR-BI activity may provide a novel target for therapeutic intervention in atherosclerotic cardiovascular disease.

Key Words: adenovirus • HDL • receptors, lipoprotein • recombinant protein

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Epidemiological data indicate a strong inverse association between HDL cholesterol levels and atherosclerotic cardiovascular disease in humans.¹ The atheroprotective effect of HDL has been postulated to be due, in part, to a process called "reverse cholesterol transport,"^{2 3} in which excess cholesterol is removed from arterial cells and returned to the liver for excretion into the bile. In reverse cholesterol transport, HDL is proposed to remove excess unesterified cholesterol from cells in the arterial intima, after which this cholesterol is esterified by lecithin:cholesteryl acyltransferase in the plasma and then transported directly or indirectly to the liver for excretion into the bile. Indirect transport to the liver involves transfer of some of the cholesteryl ester of HDL to other lipoproteins by the cholesteryl ester transfer protein (CETP² ), with subsequent receptor-mediated endocytosis of these lipoproteins by the liver. Some species (eg, mice² ) do not make CETP, and this mechanism of indirect transport is not available. Direct transport to the liver involves the binding of HDL to the surfaces of hepatocytes and efficient transfer into the cells of the cholesteryl ester but not other HDL components (eg, its protein and phospholipid outer shell). This process, which is distinct from receptor-mediated endocytosis, is called "selective cholesterol uptake"⁴ and is also used for the delivery of HDL cholesteryl ester to steroidogenic tissue.

The HDL receptor scavenger receptor class B type I (SR-BI) plays an important role in mediating selective uptake of HDL cholesterol by hepatocytes and steroidogenic cells in vitro and in vivo.^{5 6 7 8 9 10 11} For example, mice with homozygous null mutations in the SR-BI gene exhibit a 2-fold increase in plasma cholesterol. As expected for animals lacking the major receptor for selective uptake of HDL cholesterol, most of the increase was due to accumulation in HDL particles of much greater than normal size, whereas there was virtually no increase in plasma apoA-I concentration.⁷ Furthermore, mice bearing an insertion in the promoter region of the SR-BI gene that, in the homozygous state, results in 50% of normal hepatic SR-BI expression exhibit increased plasma HDL cholesterol and reduced hepatic selective uptake of HDL cholesterol.⁸ In addition, hepatic overexpression of SR-BI in mice injected with a recombinant adenovirus encoding SR-BI results in markedly reduced levels of HDL cholesterol and increased biliary cholesterol content.¹⁰ Similar results have been obtained in transgenic mice overexpressing SR-BI in the liver.^{12 13} Therefore, hepatic SR-BI may mediate one of the critical steps in reverse cholesterol transport, the direct delivery of HDL cholesteryl ester to the liver.

Because the concentration of murine plasma HDL cholesterol is inversely proportional to the level of hepatic SR-BI expression, the potential influence of SR-BI on atherogenesis has been unclear. Increased hepatic SR-BI activity may be expected to increase reverse cholesterol transport and protect against atherogenesis, yet the simultaneously decreased steady-state concentration of plasma HDL cholesterol might be proatherogenic. In addition, SR-BI can bind apoB-containing lipoproteins,¹⁴ in some circumstances can influence their plasma concentrations in vivo,^{12 13 15} and thus might influence atherosclerosis by affecting the levels of non-HDL cholesterol. To address these issues, we examined the effects of adenovirus-mediated gene transfer and transient hepatic overexpression of SR-BI on atherosclerosis in a murine model of atherosclerosis, the LDL receptor (LDLR)–deficient mouse fed a high-fat/high-cholesterol diet.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Mice and Diet
Female LDLR knockout (LDLR-deficient) mice¹⁶ crossed 10 times onto a C57BL/6 background were obtained from the Jackson Laboratory, Bar Harbor, Me. At either 2 weeks or 12 weeks before adenovirus infusion, mice were placed and maintained on a western-type diet containing 21% butterfat, 1% safflower oil, and 0.15% cholesterol¹⁷ obtained from Dyets, Inc. All procedures followed were in accordance with institutional guidelines.

Recombinant Adenoviruses
Ad.E1 and Ad.mSR-BI have been described previously.¹⁰ Briefly, Ad.mSR-BI contains an expression cassette encoding a cytomegalovirus enhancer-promoter, murine SR-BI cDNA, and an SV40 polyadenylation site inserted into the E1 region of adenovirus; Ad.E1 has the E1-region deletion with no inserted cassette. Both of the recombinant, replication-defective adenoviruses contain partial E3 deletions. Mice were injected via the tail vein with 1x10¹¹ particles of purified recombinant adenovirus in a volume of 0.1 mL. At the indicated times, blood was collected from the retro-orbital venous plexus into heparinized capillary tubes. Mice were euthanized and aortas harvested 4 weeks after adenovirus injection.

Plasma Cholesterol Assays/FPLC and Immunohistochemical Analysis of Hepatic mSR-BI Expression
The cholesterol, HDL cholesterol, apoA-I, and apoB concentrations in whole plasma were analyzed with a Cobas/Fara autoanalyzer as previously described.¹⁸ HDL cholesterol was measured with an assay kit (EZ HDL) from Sigma Diagnostics, Inc. HDL cholesterol levels in hyperlipidemic mouse plasma as assayed with this reagent were previously found to compare favorably with HDL cholesterol levels as determined by heparin manganese precipitation and by analysis of mixtures of purified (human) lipoproteins.¹⁹ Non-HDL cholesterol was determined by subtracting HDL from total cholesterol. The apoA-I and apoB assays (Sigma Diagnostics, Inc) used human standards. Fast protein liquid chromatography (FPLC) analysis was performed on individual plasma samples as previously described.^{10 18} In the 6-week feeding protocol, at 4 weeks after virus injection, mice were euthanized and livers were stained with an anti–SR-BI antibody to ensure that transgene expression was obtained in the SR-BI virus–infused mice. All of the mice injected with Ad.mSR-BI showed staining above the background (ie, endogenous) SR-BI levels at day 28 except for 1 mouse, which had no detectable increase in SR-BI expression and no changes in plasma HDL cholesterol levels. This mouse was excluded from the analysis. Livers from mice injected with Ad.E1 were also stained with a polyclonal anti-adenovirus antibody to confirm adenovirus infection.²⁰

Quantification of Atherosclerotic Lesions
Mice were anesthetized and then gently perfused with PBS via a needle placed in the left ventricle, after which the aorta was removed and fixed in 10% buffered formalin/PBS for 3 days. Adventitial and adipose tissues were removed by careful dissection, and the outer curvature of the arch was cut longitudinally. For the 6-week feeding protocol, aortas were stained with oil red O solution (1.8% oil red O, wt/vol, in 60% isopropanol, filtered twice with a 0.2-µm filter) for 15 minutes and destained with 60% isopropanol for 5 minutes to eliminate background staining.^{21 22} The arches were mounted in Aquamount on a glass slide. For the 16-week feeding protocol, the fixed aortas were briefly rinsed in 70% ethanol, stained for 6 minutes with Sudan IV (0.5% Sudan IV/35% ethanol/50% acetone), and destained for 5 minutes in 80% ethanol.²³ Because the lesions were large in volume, these aortas did not have coverslips applied but instead were pinned onto a black wax surface. Images were captured, and the red-stained lesion area was quantified with Image Pro Plus image analysis software (Media Cybernetics). Each sample was quantified independently by 2 individuals, and the results were averaged. A comparison of lesion sizes between the control group and SR-BI–treated mice was performed with the nonparametric Mann-Whitney test and Prism software (6-week experiment) and a parametric test (16-week experiment; normal distribution).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of SR-BI Overexpression on Early Atherosclerotic Lesions
Two protocols were used to evaluate the effects of SR-BI overexpression on atherosclerosis in the fat-fed LDLR-deficient mouse. First, in 2 separate experiments, female LDLR-deficient mice were placed on the western-type diet for 2 weeks, injected with recombinant adenovirus, and maintained for another 4 weeks on the diet before the harvesting of their aortas for quantification of atherosclerosis ("6-week feeding protocol"). The recombinant, replication-defective adenoviruses used were a control virus containing no transgene expression cassette (Ad.E1) and a virus encoding the murine SR-BI cDNA under the control of the cytomegalovirus enhancer/promoter (Ad.mSR-BI).¹⁰

Figure 1 shows the cholesterol/lipoprotein profiles for mice sampled immediately before virus injection (day 0) and 7 days after injection. One group of mice received the control virus (A) and the other Ad.mSR-BI (B). The plasma of uninjected LDLR-deficient mice on a western-type diet contains substantial amounts of IDL/LDL cholesterol, with lower but readily detectable amounts of VLDL and HDL cholesterol. Injection of the control virus, Ad.E1, resulted in no alterations in HDL cholesterol (Figure 1A, Table 1) or in levels of apoA-I, the major protein component of HDL (Table 1). However, injection with Ad.mSR-BI substantially decreased HDL cholesterol on postinjection days 7 (Figure 1B; Table 1) and 14 (Table 1). These results are similar to the results previously observed in wild-type mice,¹⁰ in which Ad.mSR-BI injection caused a dramatic decrease in plasma HDL cholesterol, and also demonstrate that this decrease can occur even in the presence of substantially elevated circulating levels of LDL. ApoA-I, the major protein component of HDL, was more modestly reduced in the Ad.mSR-BI–injected mice (Table 1). Although it appears that there was a substantially smaller SR-BI overexpression–mediated reduction of apoA-I levels in western diet–fed LDLR-deficient mice than in chow-fed, wild-type mice,¹⁰ these observations were made in separate experiments using different viral stocks and could be due to a variety of experimental variations (eg, precise levels of SR-BI expression) and perhaps not to the differences in genotypes and diets. The mean HDL cholesterol levels after adenovirus injection were significantly lower in the SR-BI–overexpressing mice than in control mice (Table 1, P<0.01).

View larger version (23K): [in this window] [in a new window]   Figure 1. FPLC analysis of plasma lipoproteins from western diet–fed LDLR-deficient mice. Plasma samples were drawn either before (•) or 7 days after () injection with either a control adenovirus (Ad.E1, A) or the mSR-BI–expressing adenovirus (Ad.mSR-BI, B). The samples were subjected to FPLC separation on Superose 6 columns, and each fraction was assayed for its cholesterol content as described in Methods. The first peak, fractions 4 to 8, represents VLDL; the second peak, fractions 9 to 29, IDL/LDL; and the third peak, fractions 30 to 40, HDL. Samples were run individually, and data were pooled; graphs show mean±SEM (Ad.E1, n=5; Ad.mSR-BI, n=6).

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid and Protein Concentrations in LDLR-Deficient Mice as a Function of Time After Injection of Recombinant Adenovirus in the 6-Week Feeding Protocol

Compared with changes in HDL, the amounts of non-HDL lipoproteins, which contained most of the plasma cholesterol (>90%, Figure 1 and Table 1), changed much less dramatically in the SR-BI–overexpressing mice. Triglyceride levels increased in the SR-BI–overexpressing mice on day 7 (Table 1), simultaneously with the biggest decrease in HDL cholesterol and an increase in VLDL cholesterol (Figure 1). Plasma apoB levels (Table 1), which provide a marker for non-HDL lipoproteins, were slightly decreased after injection of Ad.mSR-BI, but only on day 14 was this decrease significantly lower than that for controls (P<0.05). The effects of SR-BI overexpression on non-HDL cholesterol were also small. Seven days after injection, a modest reduction in the relative amounts of cholesterol within the largest lipoproteins was determined by FPLC (VLDL and IDL/LDL, Figure 1A and 1B) and non-HDL cholesterol determined enzymatically (Table 1), but this occurred in both control and SR-BI–overexpressing groups of mice, and the IDL/LDL size particles were somewhat smaller in both groups (shift to the right in Figure 1), presumably as a result of general effects of adenovirus injection. Although there was a trend toward lower levels of non-HDL cholesterol in the SR-BI–overexpressing mice (Figure 1B and Table 1), neither the non-HDL cholesterol levels at the individual time points nor the mean value for all time points after injection (P>0.3) were significantly different from those of controls. Taken together, these data show that transient overexpression of SR-BI resulted in a significant, although transient, decrease in HDL cholesterol and a much smaller change in non-HDL cholesterol.

To determine whether hepatic overexpression of mSR-BI affected the course of atherogenesis in the 6-week feeding protocol, we euthanized the animals (15 controls, 11 Ad.mSR-BI–injected) 4 weeks after injection and quantitatively assessed the extent of atherosclerosis using an en face assay.^{21 22 23} Figure 2A shows that the Ad.mSR-BI–injected animals exhibited significantly less lesion area than control mice (mean values of 297 366 and 86 637 µm², respectively; P=0.033). This is a remarkable result, given the apparently transient nature of SR-BI overexpression and its effects on plasma lipoproteins (see above). Particularly striking was the animal-by-animal correlation of day 7 HDL cholesterol levels with atherosclerosis measured on day 28 (not shown). Of the 7 animals with substantial atherosclerosis (lesions >300 000 µm²), only 1 had an HDL cholesterol of <100 mg/dL. Of the other animals with little atherosclerosis, only 4 of 15 had HDL cholesterol of 100 mg/dL. Thus, in this experiment, lower HDL cholesterol levels were directly correlated with decreased lesion size (P=0.0067). Furthermore, the average HDL cholesterol levels (combined mean from days 7, 14, 21, and 28) were also directly correlated with lesion size (P=0.012, Figure 3A). Although there was a weak correlation of the non-HDL cholesterol levels on day 14 with decreased lesion size (P=0.046), the combined mean non-HDL cholesterol levels over the time course did not correlate with lesion size (P=0.132; r²=0.1697). In addition, apoB levels, as well as day 7 IDL/LDL cholesterol levels, were not correlated with atherosclerotic lesion size. Taken together, these data for the effects of acute SR-BI overexpression in western diet–fed LDLR-deficient mice indicate a stronger relationship of atherosclerotic lesion size with HDL cholesterol levels than with non-HDL cholesterol levels.

View larger version (21K): [in this window] [in a new window]   Figure 2. Atherosclerotic lesion size in aortas from LDLR-deficient mice maintained on the western-type diet for 6 (A) or 16 (B) weeks. LDLR-deficient mice were maintained on the 6-week (A) or 16-week (B) western diet feeding protocol (2 or 12 weeks before and then 4 weeks after adenovirus injection), then euthanized, and the aortic arches were analyzed for the presence of oil red O–stained (6-week) or Sudan IV–stained (16-week) lesions as described in Methods. The data are expressed as average values, with error bars designating the SEM. A, Ad.E1, n=15; Ad.mSR-BI, n=11; *P=0.033. B, n=9 for each group; *P=0.034.

View larger version (17K): [in this window] [in a new window]   Figure 3. Correlation of mean HDL cholesterol levels with atherosclerotic lesion size. For both the 6-week feeding protocol (A) and the 16-week feeding protocol (B), the lesion size for each animal is shown as a function of mean HDL cholesterol level (see Tables 1 and 2). The lines represent linear regression analyses, and the r2 values for the analyses are shown.

Effects of SR-BI Overexpression on More Advanced Atherosclerotic Lesions
To verify and extend these findings, we performed similar experiments with 2 additional groups of LDLR-deficient mice receiving a modified feeding protocol. These mice were placed on the western-type diet for 12 weeks before virus injection to determine whether hepatic SR-BI overexpression would have similar effects in animals with more advanced aortic arch lesions. The mice were euthanized 4 weeks after virus injection, a total of 16 weeks on diet. Table 2 shows the effects of SR-BI overexpression on plasma total cholesterol, non-HDL cholesterol, and HDL cholesterol levels. For HDL cholesterol, the effects of SR-BI overexpression were significant for 2 of the 3 postinjection times (days 4 and 28) as well as for the overall mean, whereas only the postinjection day 28 values for total and non-HDL cholesterol were significantly different in the SR-BI–overexpressing mice (Table 2). After the mice were killed, the perfused and fixed aortas were examined visually. Lesions in the mice were sufficiently large that the extent of atherosclerosis in the unstained, intact aortas could be assessed with a dissecting microscope (Figure 4). Blind, semiquantitative estimates of unstained lesion sizes from the aortic arch through the carotid branch points by 3 observers^{21 22} showed a significant reduction in the SR-BI–overexpressing animals (P=0.017, data not shown). The lesion areas in these samples were also quantified after Sudan IV staining (see Methods) and are shown in Figure 2B. As was observed with lesions in the 6-week feeding protocol, the SR-BI–overexpressing mice exhibited significantly smaller lesions than control mice (P=0.034). The animal-by-animal correlations of atherosclerosis with mean HDL and non-HDL cholesterol levels (averaged over all postinjection measurements, including both control and SR-BI–overexpressing animals) were striking. Both the mean HDL cholesterol levels (Figure 3B) and the mean non-HDL cholesterol levels were strongly correlated with atherosclerotic lesion size (P=0.0008 and 0.0007 and r²=0.5150 and 0.5207, respectively) when both groups of mice were pooled. In addition, the mean apoA-I levels after injection in the SR-BI–injected mice were significantly lower than in those injected with control virus, 41±5 mg/dL compared with 60±6 mg/dL (P=0.02). ApoA-I levels and lesion size were also directly correlated (P=0.003; r²=0.4419).

View this table: [in this window] [in a new window]   Table 2. Plasma Lipid Concentrations as a Function of Time After Injection With Recombinant Adenovirus in the 16-Week Feeding Protocol

View larger version (62K): [in this window] [in a new window]   Figure 4. Aortic atherosclerosis in control virus–injected (A) or Ad.mSR-BI–injected (B) LDLR-deficient mice maintained on the western-type diet for 16 weeks (injected with virus after 12 weeks).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We used adenovirus-mediated gene overexpression in the livers of LDLR-deficient mice fed a western diet to assess the influence of the HDL receptor SR-BI on lipoprotein metabolism and atherosclerosis. Consistent with our previous studies in wild-type mice fed a chow diet,¹⁰ we found that overexpression of SR-BI markedly reduced HDL cholesterol levels (eg, 55% reduction 7 days after injection), indicating that transient SR-BI overexpression influences HDL metabolism even in the setting of substantially elevated non-HDL cholesterol. These results provide further support for the proposal that hepatic SR-BI is an HDL receptor that plays a role in determining circulating levels of HDL.^{5 6 7 8 9 10 13 15}

SR-BI has previously been shown to bind apoB-containing lipoproteins in vitro^{14 24} ; however, the relatively low levels of apoB-containing lipoproteins in chow-diet–fed wild-type mice made it difficult to determine the effects of SR-BI overexpression in vivo in those animals. LDLR-deficient mice fed a high-fat/high-cholesterol diet have high levels of apoB-containing plasma lipoproteins. We found in these animals that compared with its effects on HDL cholesterol, transient hepatic overexpression of SR-BI modestly reduced the levels of apoB-containing lipoprotein cholesterol (non-HDL cholesterol) and apoB itself only at some time points after Ad.mSR-BI injection and did not have significant effects on the mean levels. Decreased non-HDL cholesterol levels due to SR-BI overexpression have been reported for SR-BI transgenic mice.^{12 13 15} Additional work will be necessary to determine whether the decreases in the apoB-containing lipoproteins were due to direct interactions of these lipoproteins with SR-BI or were indirect consequences of SR-BI overexpression (eg, secondary consequences of changes in plasma HDL). Our results indicate that transient overexpression of SR-BI via gene transfer has a much less pronounced effect on apoB-containing lipoprotein cholesterol levels in LDLR-deficient mice than does transgenic overexpression,^{12 13} suggesting that the effects of hepatic SR-BI overexpression on the metabolism of apoB-containing lipoproteins may be more complex than currently appreciated.

Strikingly, we found that transient hepatic overexpression of SR-BI significantly reduced atherosclerosis in western diet–fed LDLR-deficient mice at 2 different times of lesion development. Although additional studies will be necessary to define the molecular basis for the protective effects of SR-BI, several potentially interrelated mechanisms may account for this result. First, hepatic overexpression of SR-BI is expected to substantially increase the flux of HDL cholesterol through the reverse cholesterol transport pathway from the arterial wall to the liver and then out of the body via the bile.^{10 12 15 19} As a consequence, the rate of plaque deposition would be expected to decrease, and plaque regression might even occur. Second, a consequence of hepatic SR-BI overexpression may be the generation of altered HDLs with increased antiatherogenic properties (eg, enhanced cellular cholesterol efflux²⁵ and/or greater access to the arterial intima²⁶ ). Third, hepatic overexpression of SR-BI might alter the amounts or structures of non-HDL lipoproteins (eg, apoB-containing lipoproteins) and thus diminish their atherogenicity. Our analysis of the correlations between lipoprotein component levels and lesion sizes, especially HDL cholesterol levels (see Results), suggests that in this system, changes in HDL cholesterol levels, presumably due to increased flux through the reverse cholesterol transport pathway, are likely to have had a major impact on atherosclerosis. Although there was a trend toward reduced levels of non-HDL cholesterol in the SR-BI–overexpressing animals compared with controls, substantial animal-to-animal variation resulted in no statistically significant SR-BI overexpression–dependent differences in 1 of the 2 experimental protocols (6-week protocol).

Nevertheless, it is possible that changes in non-HDL lipoproteins influenced atherosclerosis in this study. In the 16-week protocol, there were strong correlations between lesion size of each animal and both mean HDL cholesterol and mean non-HDL cholesterol levels of each animal. Arai et al¹⁵ reported studies of the effects on atherosclerosis of hepatic SR-BI overexpression in SR-BI transgenic mice bred into a heterozygous LDLR-deficient background. When these mice were fed a 1.25% cholesterol/cholic acid (Paigen) diet, the SR-BI transgenic mice developed less atherosclerosis than controls and had lower levels of HDL and non-HDL cholesterol. There are, however, a number of differences between the results of Arai et al and those reported here. In contrast to their results with the Paigen diet, when they fed mice a western-type diet similar to the one used here, they did not observe significant differences in non-HDL cholesterol levels or atherosclerotic lesion sizes in SR-BI–overexpressing and control animals. Furthermore, they found a strong correlation of atherosclerosis with non-HDL cholesterol but little correlation with HDL cholesterol, whereas we found a strong HDL cholesterol/lesion size correlation. Finally, it is possible that some of the differences in these studies arise because of different effects of chronic versus transient expression, different levels of hepatic SR-BI overexpression, or differences in the timing of SR-BI overexpression relative to the atherosclerotic disease process.

The results reported here represent another example of the positive association of HDL cholesterol levels with atherosclerosis. Previous reports from other laboratories have shown that transgenic overexpression of CETP reduces both HDL cholesterol levels and atherosclerosis in mice²⁷ and that a deficiency of CETP is associated with increases in both HDL cholesterol levels and risk of coronary heart disease in humans.²⁸ Furthermore, transgenic overexpression of lecithin:cholesteryl acyltransferase increases both HDL cholesterol and atherosclerosis in mice.²⁹ When considered with the antiatherogenic effects of apoA-I overexpression in mice^{30 31} but only modest proatherogenic effects of reduced apoA-I expression^{32 33} and the well-known inverse correlation of plasma HDL cholesterol levels and atherosclerosis in humans,¹ these data highlight the complex interplay of lipoproteins, receptors, and plasma-modifying enzymes with atherogenesis. The influence of decreasing or increasing HDL cholesterol levels on the risk for atherosclerosis clearly depends on the mechanisms by which the HDL cholesterol levels are modulated and thus may dramatically affect potential antiatherosclerosis therapies focused on HDL cholesterol levels.

In summary, adenovirus-mediated overexpression of murine SR-BI in LDLR-deficient mice fed a western-type diet significantly reduced atherosclerosis despite markedly reducing HDL cholesterol levels and only modestly lowering non-HDL cholesterol levels. This suggests that hepatic SR-BI expression can influence the rate of selective HDL cholesterol uptake in the liver and raises the possibility that hepatic SR-BI expression can also influence the overall rate of reverse cholesterol transport from arterial wall to liver. If so, these data would suggest that manipulation of the rate of reverse cholesterol transport by altering expression of an HDL receptor in vivo can have a positive impact on atherogenesis. Consistent with the results reported here are recent studies that have shown that elimination of SR-BI activity in apoE-deficient mice raises HDL cholesterol levels and dramatically accelerates atherogenesis.¹⁹ Furthermore, the data reported here demonstrate that intervention via hepatic SR-BI overexpression after the establishment of either early or more advanced atherosclerotic lesions is effective at reducing the progression of murine atherosclerosis. Because the tissue distribution and regulation of expression of SR-BI in humans or cultured human cells resembles that in mice,^{11 34 35 36} these findings raise the possibility that intervention targeted toward increasing hepatic SR-BI expression might provide a novel approach to the prevention and treatment of atherosclerotic cardiovascular disease.

	Acknowledgments

 
This work was supported by grant HL-55323 from the National Institutes of Health and a grant from the W.W. Smith Foundation to D.J. Rader and grant HL-41484 from the National Institutes of Health, Heart, Lung, and Blood Institute, to M. Krieger. We would like to thank Pearle Smith, Anna Lillethun, Karl Kozarsky, and Ivan Liachko for expert technical assistance.

Received July 19, 1999; accepted October 19, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Gordon DJ, Rifkind BM. High-density lipoproteins: the clinical implications of recent studies. N Engl J Med. 1989;321:1311–1316.[Medline] [Order article via Infotrieve]
2. Tall AR. Plasma high density lipoproteins: metabolism and relationship to atherogenesis. J Clin Invest. 1990;86:379–384.
3. Reichl D, Miller NE. Pathophysiology of reverse cholesterol transport: insights from inherited disorders of lipoprotein metabolism. Arteriosclerosis. 1989;9:785–797.[Free Full Text]
4. Glass C, Pittman RC, Weinstein DB, Steinberg D. Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad. Proc Natl Acad Sci U S A. 1983;80:5435–5439.[Abstract/Free Full Text]
5. Acton S, Rigotti A, Landschultz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996;271:460–461.[Medline] [Order article via Infotrieve]
6. Landschultz KT, Pathak RK, Rigotti A, Krieger M, Hobbs HH. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat. J Clin Invest. 1996;98:984–995.[Medline] [Order article via Infotrieve]
7. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key in HDL metabolism. Proc Natl Acad Sci U S A. 1997;94:12610–12615.[Abstract/Free Full Text]
8. Varban ML, Rinninger F, Wang N, Fairchild-Huntress V, Dunmore JH, Fang Q, Gosselin ML, Dixon KL, Deeds JD, Acton SL, Tall AR, Huszar D. Targeted mutation reveals a central role for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol. Proc Natl Acad Sci U S A. 1998;95:4619–4624.[Abstract/Free Full Text]
9. Krieger M. The "best" of cholesterols, the "worst" of cholesterols: a tale of two receptors. Proc Natl Acad Sci U S A. 1998;95:4077–4080.[Free Full Text]
10. Kozarsky KF, Donahee MH, Rigotti A, Iqbal S, Edelman ER, Krieger M. Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels. Nature. 1997;387:414–417.[Medline] [Order article via Infotrieve]
11. Krieger M. Charting the fate of the "good cholesterol": identification and characterization of the high-density lipoprotein receptor SR-BI. Annu Rev Biochem. 1999;68:523–558.[Medline] [Order article via Infotrieve]
12. Wang N, Arai T, Ji Y, Rinninger F, Tall AR. Liver-specific overexpression of scavenger receptor BI decreases levels of very low density lipoprotein apoB, low density lipoprotein apoB, and high density lipoprotein in transgenic mice. J Biol Chem. 1998;273:32920–32926.[Abstract/Free Full Text]
13. Ueda Y, Royer L, Gong E, Zhang J, Cooper PN, Francone O, Rubin EM. Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice. J Biol Chem. 1999;274:7165–7171.[Abstract/Free Full Text]
14. Acton SL, Scherer PE, Lodish HF, Krieger M. Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem. 1994;269:21003–21009.[Abstract/Free Full Text]
15. Arai T, Wang N, Bezouevski M, Welch C, Tall AR. Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene. J Biol Chem. 1999;274:2366–2371.[Abstract/Free Full Text]
16. Ishibashi S, Goldstein J, Brown M, Herz J, Burns D. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest. 1994;93:1885–1893.
17. Breslow JL. Mouse models of atherosclerosis. Science. 1996;272:685–688.[Abstract]
18. Tsukamoto K, Jiester KG, Smith P, Usher DC, Glick JM, Rader DJ. Comparison of human apoA-I expression in mouse models of atherosclerosis after gene transfer using a second generation adenovirus. J Lipid Res. 1997;38:1869–1876.[Abstract]
19. Ye X, Robinson M, Batshaw M, Furth E, Smith I, Wilson JM. Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors. J Biol Chem. 1996;271:3639–3646.[Abstract/Free Full Text]
20. Xiao Q, Danton MJS, Witte DP, Kowala MC, Valentine MT, Degen JL. Fibrinogen deficiency is compatible with the development of atherosclerosis in mice. J Clin Invest. 1998;101:1184–1194.[Medline] [Order article via Infotrieve]
21. Xiao Q, Danton MJS, Witte DP, Kowala MC, Valentine MT, Bugge TH, Degen JL. Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis. Proc Natl Acad Sci U S A. 1997;94:10335–10340.[Abstract/Free Full Text]
22. Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J Lipid Res. 1995;36:2320–2328.[Abstract]
23. Calvo D, Gomez-Corondado D, Lasuncion MA, Vega MA. CLA-1 is an 85-kD plasma membrane glycoprotein that acts as a high-affinity receptor for both native (HDL, LDL, and VLDL) and modified (OxLDL and AcLDL) lipoproteins. Arterioscler Thromb Vasc Biol. 1997;17:2341–2349.[Abstract/Free Full Text]
24. Francone OL, Fielding CJ, Fielding PE. Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques. J Lipid Res. 1990;31:2195–2200.[Abstract]
25. Reichl D. Extravascular circulation of lipoproteins: their role in reverse transport of cholesterol. Atherosclerosis. 1994;105:117–129.[Medline] [Order article via Infotrieve]
26. Hayek T, Masucci-Magoulas L, Jiang X, Walsh A, Rubin E, Breslow JL, Tall AR. Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene. J Clin Invest. 1995;96:2071–2074.
27. Zhong S, Sharp DS, Grove JS, Bruce C, Yano K, Curb JD, Tall AR. Increased coronary heart disease in Japanese-American men with mutations in the cholesteryl ester transfer protein gene despite increased HDL levels. J Clin Invest. 1996;97:2917–2923.[Medline] [Order article via Infotrieve]
28. Berard AM, Foger B, Remaley A, Shamburek R, Vaisman BL, Talley G, Paigen B, Hoyt RF Jr, Marcovina S, Brewer HB Jr, Santamarina-Fojo S. High plasma HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin:cholesteryl acyltransferase. Nat Med. 1997;3:744–749.[Medline] [Order article via Infotrieve]
29. Plump A, Scott C, Breslow J. Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse. Proc Natl Acad Sci U S A. 1994;91:9607–9611.[Abstract/Free Full Text]
30. Paszty C, Maeda N, Verstuyft J, Rubin EM. Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J Clin Invest. 1994;94:899–903.
31. Voyiaziakis E, Goldberg IJ, Plump AS, Rubin EM, Breslow JL, Huang LS. ApoA-I deficiency causes both hypertriglyceridemia and increased atherosclerosis in human apoB transgenic mice. J Lipid Res. 1998;39:313–321.[Abstract/Free Full Text]
32. Hughes SD, Verstuyft J, Rubin EM. HDL deficiency in genetically engineered mice requires elevated LDL to accelerate atherogenesis. Arterioscler Thromb Vasc Biol. 1997;17:1725–1729.[Abstract/Free Full Text]
33. Trigatti B, Rayburn H, Vinals M, Braun A, Miettinen H, Penman M, Hertz M, Schrenzel M, Amigo L, Rigotti A, Krieger M. Influence of the HDL receptor SR-BI on reproductive and cardiovascular pathophysiology. Proc Natl Acad Sci U S A. 1999;6:9322–9327.
34. Calvo D, Vega MA. Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J Biol Chem. 1993;268:18929–18935.[Abstract/Free Full Text]
35. Murao K, Terpstra V, Green SR, Kondratenko N, Steinberg D, Quehenberger O. Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes. J Biol Chem. 1997;272:17551–17557.[Abstract/Free Full Text]
36. Cao G, Garcia CK, Wyne KL, Schultz RA, Parker KL, Hobbs HH. Structure and localization of the human gene encoding SR-BI/CLA-1: evidence for transcriptional control by steroidogenic factor 1. J Biol Chem. 1997;272:33068–33076.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Komori, H. Arai, T. Kashima, T. Huby, T. Kita, and Y. Ueda Coexpression of CLA-1 and Human PDZK1 in Murine Liver Modulates HDL Cholesterol Metabolism Arterioscler. Thromb. Vasc. Biol., July 1, 2008; 28(7): 1298 - 1303. [Abstract] [Full Text] [PDF]

				M. Z. Ashraf, N. S. Kar, X. Chen, J. Choi, R. G. Salomon, M. Febbraio, and E. A. Podrez Specific Oxidized Phospholipids Inhibit Scavenger Receptor BI-mediated Selective Uptake of Cholesteryl Esters J. Biol. Chem., April 18, 2008; 283(16): 10408 - 10414. [Abstract] [Full Text] [PDF]

				U. J. F. Tietge, N. Nijstad, R. Havinga, J. F. W. Baller, F. H. van der Sluijs, V. W. Bloks, T. Gautier, and F. Kuipers Secretory phospholipase A2 increases SR-BI-mediated selective uptake from HDL but not biliary cholesterol secretion J. Lipid Res., March 1, 2008; 49(3): 563 - 571. [Abstract] [Full Text] [PDF]

				M. Van Eck, M. Hoekstra, R. Out, I. S. T. Bos, J. K. Kruijt, R. B. Hildebrand, and T. J. C. Van Berkel Scavenger receptor BI facilitates the metabolism of VLDL lipoproteins in vivo J. Lipid Res., January 1, 2008; 49(1): 136 - 146. [Abstract] [Full Text] [PDF]

				T. J. F. Nieland, J. T. Shaw, F. A. Jaipuri, Z. Maliga, J. L. Duffner, A. N. Koehler, and M. Krieger Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI J. Lipid Res., August 1, 2007; 48(8): 1832 - 1845. [Abstract] [Full Text] [PDF]

				J. C. Wu, F. M. Bengel, and S. S. Gambhir Cardiovascular Molecular Imaging Radiology, August 1, 2007; 244(2): 337 - 355. [Abstract] [Full Text] [PDF]

				J.-Y. Lee, R. M. Badeau, A. Mulya, E. Boudyguina, A. K. Gebre, T. L. Smith, and J. S. Parks Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice J. Lipid Res., May 1, 2007; 48(5): 1052 - 1061. [Abstract] [Full Text] [PDF]

				C. D. Akpovi, S. R. Yoon, M. L. Vitale, and R-M. Pelletier The predominance of one of the SR-BI isoforms is associated with increased esterified cholesterol levels not apoptosis in mink testis J. Lipid Res., October 1, 2006; 47(10): 2233 - 2247. [Abstract] [Full Text] [PDF]

				A. Yesilaltay, O. Kocher, R. Pal, A. Leiva, V. Quinones, A. Rigotti, and M. Krieger PDZK1 Is Required for Maintaining Hepatic Scavenger Receptor, Class B, Type I (SR-BI) Steady State Levels but Not Its Surface Localization or Function J. Biol. Chem., September 29, 2006; 281(39): 28975 - 28980. [Abstract] [Full Text] [PDF]

				K. J. Moore and M. W. Freeman Scavenger Receptors in Atherosclerosis: Beyond Lipid Uptake Arterioscler. Thromb. Vasc. Biol., August 1, 2006; 26(8): 1702 - 1711. [Abstract] [Full Text] [PDF]

				K. Kitajima, D. H.L. Marchadier, G. C. Miller, G.-p. Gao, J. M. Wilson, and D. J. Rader Complete Prevention of Atherosclerosis in ApoE-Deficient Mice by Hepatic Human ApoE Gene Transfer With Adeno-Associated Virus Serotypes 7 and 8 Arterioscler. Thromb. Vasc. Biol., August 1, 2006; 26(8): 1852 - 1857. [Abstract] [Full Text] [PDF]

				X.-A. Li, L. Guo, R. Asmis, M. Nikolova-Karakashian, and E. J. Smart Scavenger Receptor BI Prevents Nitric Oxide-Induced Cytotoxicity and Endotoxin-Induced Death Circ. Res., April 14, 2006; 98(7): e60 - e65. [Abstract] [Full Text] [PDF]

				P. C.N. Rensen, L. A.J.M. Sliedregt, P. J. van Santbrink, M. Ferns, H. N.J. Schifferstein, S. H. van Leeuwen, J. H.M. Souverijn, T. J.C. van Berkel, and E. A.L. Biessen Stimulation of Liver-Directed Cholesterol Flux in Mice by Novel N-Acetylgalactosamine-Terminated Glycolipids With High Affinity for the Asialoglycoprotein Receptor Arterioscler. Thromb. Vasc. Biol., January 1, 2006; 26(1): 169 - 175. [Abstract] [Full Text] [PDF]

				R. Out, M. Hoekstra, S. C. A. de Jager, P. de Vos, D. R. van der Westhuyzen, N. R. Webb, M. Van Eck, E. A. L. Biessen, and T. J. C. Van Berkel Adenovirus-mediated hepatic overexpression of scavenger receptor class B type I accelerates chylomicron metabolism in C57BL/6J mice J. Lipid Res., June 1, 2005; 46(6): 1172 - 1181. [Abstract] [Full Text] [PDF]

				D. R. Greaves and S. Gordon Thematic review series: The Immune System and Atherogenesis. Recent insights into the biology of macrophage scavenger receptors J. Lipid Res., January 1, 2005; 46(1): 11 - 20. [Abstract] [Full Text] [PDF]

				A. J. Merched and L. Chan Absence of p21Waf1/Cip1/Sdi1 Modulates Macrophage Differentiation and Inflammatory Response and Protects Against Atherosclerosis Circulation, December 21, 2004; 110(25): 3830 - 3841. [Abstract] [Full Text] [PDF]

				M. Van Eck, I. S. T. Bos, R. B. Hildebrand, B. T. Van Rij, and T. J.C. Van Berkel Dual Role for Scavenger Receptor Class B, Type I on Bone Marrow-Derived Cells in Atherosclerotic Lesion Development Am. J. Pathol., September 1, 2004; 165(3): 785 - 794. [Abstract] [Full Text] [PDF]

				T. Fu, D. Mukhopadhyay, N. O. Davidson, and J. Borensztajn 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 J. Biol. Chem., July 2, 2004; 279(27): 28662 - 28669. [Abstract] [Full Text] [PDF]

				T. J. F. Nieland, A. Chroni, M. L. Fitzgerald, Z. Maliga, V. I. Zannis, T. Kirchhausen, and M. Krieger Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide J. Lipid Res., July 1, 2004; 45(7): 1256 - 1265. [Abstract] [Full Text] [PDF]

A. M. Gotto Jr and E. A. Brinton Assessing low levels of high-density lipoprotein cholesterol as a risk factor in coronary heart disease: A working group report and update J. Am. Coll. Cardiol., March 3, 2004; 43(5): 717 - 724.