Isoform-Dependent Cholesterol Efflux From Macrophages by Apolipoprotein E Is Modulated by Cell Surface Proteoglycans

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression.

Methods and Results— When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7-ß-D-xyloside (ß-DX) resulted in all 3 isoforms’ reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with ß-DX resulted in all 3 exogenously derived apoE isoforms’ reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4.

Conclusions— The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.

Key Words: apolipoprotein E  isoforms  cholesterol efflux RAW264.7 cells  heparan-sulfate proteoglycans

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Macrophages in the arterial wall play a crucial role in the progression of atherosclerosis.¹ Macrophages are transformed to foam cells by their uptake cholesterol, form fatty streak lesions, and secrete cytokines that trigger the development of advanced complex atherosclerotic lesions.¹ Cholesterol efflux is a pivotal mechanism that reduces the accumulation of cholesterol in macrophages and in which HDL has been proposed to play an important role.²

Apolipoprotein E (apoE) is a glycoprotein produced not only by the liver but also from macrophages.^3,4 Besides its classic role in lipoprotein metabolism as a ligand for lipoprotein receptors,^3,5 several studies have suggested additional roles for apoE in the protection against atherosclerosis. Overexpression of apoE in the liver of atherosclerosis-prone mice reduced atherosclerosis, accompanied by the deposition of hepatic-derived apoE in the atherosclerotic lesions.^6,7 Macrophage-specific expression of apoE in apoE-deficient mice resulted in reduced atherosclerosis,⁸ and bone marrow transplantation from apoE-deficient mice to normal mice increased atherosclerotic lesions⁹ without affecting plasma lipid profiles. Mice overexpressing apoE in the arterial wall show reduced atherosclerotic lesions.¹⁰ These studies suggest that both macrophage-derived and liver-derived apoE possess direct antiatherogenic properties.

There are 3 major isoforms in human apoE: apoE2, E3, and E4. Many studies have clarified differences among these isoforms in terms of their effects on lipoprotein metabolism and atherosclerosis.^7,11–16 However, few studies have been performed to prove a differential effect of these isoforms on cholesterol-loaded macrophages. Cullen et al¹⁷ clarified a differential effect on cholesterol homeostasis in macrophages by apoE isoforms by using monocytes from humans homozygous for these isoforms. On the other hand, Smith et al¹⁸ did not find any difference in the efficiency for cholesterol efflux among these isoforms in RAW264.7 cells engineered to stably express the apoE isoform. Furthermore, no study has been performed to examine the isoform-specific cholesterol efflux by exogenously derived apoE under condition of nonendogenous apoE production in macrophages.

To address apoE isoform-specific effects on cholesterol metabolism in macrophages, we used adenovirus-mediated gene transfer to attain the same level of expression among the apoE isoforms. We also used 4-methylumbelliferyl 7-ß-D-xyloside (ß-DX)^19,20 to inhibit the cellular production of cell-surface heparan sulfate proteoglycans (HSPGs) that would modulate the cholesterol-effluxing ability of apoE.^21–23

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
RAW264.7 cells and HeLa cells were purchased from American Type Culture Collection (Manassas, Va) and the Health Science Research Resources Bank (Osaka, Japan), respectively. Adenoviral vectors AdE2, AdE3, and AdE4,¹⁴ encoding apoE2, E3, and E4 isoform cDNA, respectively, were used to express human apoE isoforms. AdLacZ¹⁴ was used as a control adenovirus. Acetylated LDL was prepared from normal human LDL by following a method previously described.²⁴

Determination of Multiplicity of Infection for Efficient Gene Delivery
RAW264.7 cells were infected with either 0, 80, or 240 multiplicity of infection (MOI) of AdLacZ, and HeLa cells were infected with either 0, 1, or 100 MOI of AdLacZ. Forty-eight hours after infection, cells were fixed with 0.5% glutaraldehyde and incubated overnight at 37°C with a solution containing 5 mmol/L K₃Fe(CN)₆, 5 mmol/L K₄Fe(CN)₆, 1 mmol/L MgCl₂, and 1 mg/mL 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-gal).

Expression of ApoE Isoforms in RAW264.7 Cells by Adenoviral Vectors
RAW264.7 cells were cultured with or without 1 mmol/L ß-DX; infected with 240 MOI of AdE2, AdE3, AdE4, or AdLacZ; and cholesterol-loaded with 100 µg/mL protein of acetylated LDL. Twenty-four hours later, cells were washed 3 times with PBS and cultured further for 48 hours in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.3% bovine serum albumin (BSA), and 0.3 mmol/L 8-Br-cAMP, with or without 1 mmol/L ß-DX. For Western blot analysis, cells lysed in sample buffer and cell medium were stocked at -80°C. For Northern blotting, 25 µg of cellular total RNA purified by the acid guanidinium-phenol-chloroform method was electrophoresed and blotted to nylon membranes, which were then hybridized with digoxigenin-labeled apoE3 cDNA probe followed by incubation with alkaline phosphatase-conjugated anti-digoxigenin antibody. The signals were detected with a chemiluminescence method, and the intensities of bands were quantified by using NIH Image software. After stripping the apoE probes, the membranes were reprobed with mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.

Studies With Endogenous ApoE Expression
RAW264.7 cells, cultured with or without 1 mmol/L ß-DX for 48 hours, were either infected with 240 MOI of each adenoviral vector or left uninfected with an adenoviral vector and cholesterol-loaded with 100 µg/mL protein of acetylated LDL. For cholesterol-unloaded control cells, RAW264.7 cells were neither cholesterol-loaded nor infected with adenovirus. After another 24 hours, the cells were washed 3 times with PBS and cultured for an additional 36 hours in DMEM supplemented with 0.3% BSA and 0.3 mmol/L 8-Br-cAMP, with or without 1 mmol/L ß-DX, after which lipid extraction from cells was performed as described below. To evaluate the effect of 8-Br-cAMP on cholesterol efflux, some plates were cultured without 8-Br-cAMP and ß-DX.

Studies With Exogenous ApoE
HeLa cells were either uninfected or infected with 100 MOI of each adenoviral vector. Twenty-four hours later, cells were washed 3 times with PBS and cultured for another 48 hours in DMEM supplemented with 0.3% BSA. The medium was then harvested, quickly centrifuged, and diluted with DMEM to make the final apoE concentration 50 µg/mL. The medium harvested from cells treated with AdLacZ or no adenovirus was diluted at the average ratio of those of apoE isoforms. The medium was stocked at 4°C for up to 3 days until use in the experiments described below. Aliquots of the medium were subjected to fast protein liquid chromatography (FPLC) on a Superose 6 column (Pharmacia LKB Biotechnology), and 2 adjacent fractions were pooled together and subjected to Western blot analysis.

RAW264.7 cells were cultured with or without 1 mmol/L ß-DX, and 48 hours later, 100 µg/mL acetylated LDL was added. After another 24 hours, the cells were washed 3 times with PBS and cultured in the medium obtained from HeLa cells in the presence of 0.3 mmol/L 8-Br-cAMP and 0.3% BSA, with or without 1 mmol/L ß-DX. Some plates were cultured in the absence of 8-Br-cAMP and ß-DX to assess the role of 8-Br-cAMP in cholesterol efflux. Thereafter, for the measurement of cellular lipid contents, cells were cultured for 36 hours. For the detection of cell surface-bound apoE, the cells were then cultured for 24 hours, washed 3 times with PBS, and incubated for another 4 hours with 0.5 mL DMEM with or without the addition of 2 U/mL heparinase I. The medium was quickly centrifuged, and the supernatant was preserved for Western blot analysis. The cells were harvested, and the cellular protein concentration was measured by the Lowry method.

Extraction and Quantification of Lipid From Cells
Cells were collected into tubes and washed 3 times with PBS. After a hexane/isopropanol mixture (3:2, vol/vol) was added, the tubes were vortexed and placed at room temperature for 30 minutes. After centrifugation, the supernatant was transferred to glass tubes and dried under nitrogen gas, followed by the measurement of cholesterol content; the pellet was used for measurement of cellular protein content. Total cholesterol (TC) and free cholesterol (FC) were measured by enzymatic methods, and protein concentration was measured by the Lowry method. After the adjustment of cellular TC and FC contents for cellular protein, the cellular esterified cholesterol (EC) content was calculated by subtracting the FC content value from the TC content value.

Western Blotting of ApoE
The medium collected from RAW264.7 cells, adjusted for the protein concentration of the cells, or aliquots of FPLC fractions were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were blotted to nitrocellulose membranes and reacted with goat anti-human apoE polyclonal antibody, followed by incubation with peroxidase-labeled rabbit anti-goat IgG antibody. The protein bands were visualized with 4-chloro-1-naphthol as the substrate.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Determination of MOI for Efficient Gene Delivery
The efficiency of gene delivery with adenoviral vectors was determined by X-gal staining of cells infected with AdLacZ. In RAW264.7 cells, a few faintly stained cells were observed even at an MOI of 0, owing to the endogenous activity of galactosidase. As MOI increased, the proportion of stained cells increased and the intensity of the staining became stronger. At an MOI of 240, maximal gene expression (70%) was observed. In HeLa cells, 100% of staining was observed at an MOI of 100. In consequence, for the subsequent experiments, an MOI of 240 and 100 was used for RAW264.7 cells and HeLa cells, respectively.

fig.ommitted  X-gal staining of RAW264.7 and HeLa cells. RAW264.7 cells and HeLa cells were infected with AdLacZ at different MOIs and subjected to X-gal staining. A, B, and C: RAW264.7 cells; D, E, and F: HeLa cells. The values of MOI are indicated in parentheses.

Expression of ApoE Isoforms in RAW264.7 Cells With Adenoviral Vectors
To examine whether infection at the same MOI of adenovirus resulted in the same level of expression, total RNA purified from RAW264.7 cells infected with either AdE2, E3, or E4 was subjected to Northern blot analysis . The relative ratios of apoE mRNA to GAPDH mRNA were 1.12, 1.00, and 1.02 for apoE2, apoE3, and apoE4, respectively.

fig.ommitted  A: Northern blot analysis of RAW264.7 cells infected with apoE adenoviruses. Twenty-five micrograms of total cellular RNA separated on a gel and blotted to a nylon membrane was probed with human apoE cDNA or mouse GAPDH cDNA. B, C: Western blot analysis of apoE protein from the medium harvested from RAW264.7 cells infected with apoE adenoviruses with (B) or without (C) ß-DX. LacZ, E2, E3, E4 indicate samples from cells infected with AdLacZ, AdE2, AdE3, and AdE4, respectively.

Western blot analysis of the medium revealed no difference in the protein levels among the apoE isoforms in the presence of ß-DX . However, without the addition of ß-DX, little difference was observed in protein levels among the isoforms  the relative apoE protein levels in the medium of apoE2 and apoE4 versus apoE3 were 1.93 and 1.31, respectively.

Effect of Endogenous ApoE Expression on Cellular Cholesterol
The cholesterol loading of RAW264.7 cells resulted in a 2- to 3-fold increase in cellular TC, FC, and EC contents In the absence of ß-DX, a significant reduction in the cellular TC and EC content was attained in cells expressing apoE2 (12.4% and 44.9% reduction for TC and EC, respectively, compared with LacZ). ApoE3 reduced cellular cholesterol content to a lesser degree than did apoE2, which was not significantly different from control. ApoE4 did not result in the reduction of cellular cholesterol. In the absence of 8-Br-cAMP, no change in cellular cholesterol was observed in cells expressing any apoE isoform (please see Figure IA at http:atvb.ahajournals.org). Interestingly, in the presence of ß-DX, all 3 apoE isoforms resulted in a significant reduction of cellular TC and EC contents Percentages for these reductions compared with LacZ control were as follows: 25.6% and 22.5% for TC and EC of apoE2, 33.2% and 43.1% for TC and EC of apoE3, and 28.7% and 40.6% for TC and EC of apoE4. In the presence of ß-DX, the extent of reduction in cellular cholesterol was greatest with apoE3, followed by apoE4, and least in apoE2.

fig.ommitted  Cholesterol efflux by endogenous apoE. RAW264.7 cells were infected with adenoviral vectors and loaded with cholesterol, after which lipid extraction was performed. A and B represent the results without and with treatment of the cells by ß-DX, respectively. N indicates cells untreated with acetylated LDL and adenovirus. C, Cholesterol-loaded cells untreated with adenovirus. LacZ, E2, E3, E4 indicate cholesterol-loaded cells treated with AdLacZ, AdE2, AdE3, and AdE4, respectively. Closed bars represent TC/cell protein, stippled bars represent FC/cell protein, and open bars represent EC/cell protein. Data are mean±SEM (n=6). P<0.05 compared with both C and LacZ; P<0.05 compared with E2.

Effect of Exogenous ApoE on Cellular Cholesterol
FPLC analysis of the medium from HeLa cells infected with apoE adenovirus revealed that the apoE protein, regardless of its isoform, distributed mostly on HDL and VHDL fractions and, to a lesser extent, on lipid-poor fractions 

fig.ommitted    Distribution of apoE protein on lipoprotein particles in the medium harvested from HeLa cells infected with apoE adenovirus. E2, E3, E4 indicate samples harvested from HeLa cells infected with AdE2, AdE3, and AdE4, respectively. Lanes 4 through 6 correspond to VLDL fractions, lanes 7 through 11 to IDL/LDL fractions, lanes 12 through 19 do HDL fractions, and lanes 20 through 23 to lipid-poor fractions.

In the absence of ß-DX, apoE2- and E3-containing media significantly reduced cellular TC and EC contents (22.9% or 16.9% reduction for TC and 29.1% or 20.5% reduction for EC, respectively, compared with LacZ control). Medium containing apoE4 showed trends toward a reduction of TC and EC contents; however, no significant reduction was observed. When we compared the efficiency in the reduction of TC and EC among the 3 isoforms, apoE4 was less effective than apoE2 and E3, and apoE2 showed a tendency in the increased reduction for TC and EC compared with apoE3. The reduction in cellular cholesterol by apoE isoforms was not observed in the absence of 8-Br-cAMP (please see Figure IB at http://atvb.ahajournals.org).

fig.ommitted  Cholesterol efflux by exogenous apoE. RAW264.7 cells were loaded with cholesterol by acetylated LDL and cultured in the medium containing each apoE isoform, after which lipid extraction was performed. A and B represent the results without and with treatment of the cells by ß-DX, respectively. N indicates cells untreated with acetylated LDL. C, LacZ, E2, E3, E4 indicate cholesterol-loaded cells treated with the medium harvested from HeLa cells uninfected with adenovirus or infected with AdLacZ, AdE2, AdE3, and AdE4, respectively. Closed bars represent TC/cell protein, stippled bars represent FC/cell protein, and open bars represent EC/cell protein. Data are mean±SEM (n=6). P<0.05 compared with both C and LacZ; P<0.05 compared with E4.

In the presence of ß-DX, all 3 exogenous apoE isoforms resulted in a significant reduction of cellular TC and EC contents , which was the case with endogenous apoE isoforms. However, the differential effect among the isoforms was not found in this experiment with exogenous apoE.

Detection of Cell Surface-Bound ApoE by Using Heparinase
To elucidate whether the binding ability to cell surface HSPGs differs among apoE isoforms, RAW264.7 cells were incubated with the medium harvested from HeLa cells for 24 hours, which were then washed and incubated with 2 U/mL heparinase I. The catalyzing activity of heparinase I for HSPGs was confirmed by additional experiments with lactoferrin (data not shown). In the absence of ß-DX, the amount of apoE released by heparinase I increased in the following order: E2<E3<E4 . On the other hand, when the production of cell surface HSPGs was inhibited by ß-DX, release of apoE protein from the cell surface by heparinase I was not observed

fig.ommitted  Release of apoE protein from the cell surface after treatment of RAW264.7 cells with heparinase I. A, Representative Western blotting analysis of the medium harvested from cells cultured without ß-DX. - Indicates samples untreated with heparinase I; +, samples treated with heparinase I. B, Three independent experiments from A were subjected to densitometric analysis, and the increase in the intensity of the band from (-) to (+) was measured. The relative increment for apoE2 and E4 was calculated after setting the increment in apoE3 as 1. Data are mean±SEM. P<0.05 compared with apoE2; P<0.01 compared with apoE2. C, Representative Western blotting analysis of the medium harvested from cells cultured with ß-DX. - Indicates samples untreated with heparinase I; +, samples treated with heparinase I.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
It has been difficult to induce macrophage cell lines to express any genes by the DNA transfection method. Adenoviral vectors are good tools for gene transfer into any cells that express specific integrins²⁵ and the coxsackievirus and adenovirus receptor.²⁶ Another benefit of using adenoviral vectors for gene transfer is that it is possible to make the level of expression similar among different genes by using the same promoter and infecting viruses at the same MOI. As was shown in Results, good efficiency of infection up to 70% was obtained by adenovirus-mediated gene transfer to RAW264.7 cells, and infection at the same MOI resulted in the same levels of expression among the 3 apoE isoforms. The levels of apoE protein expression analyzed with the cellular proteins were almost the same among the 3 isoforms, confirming the same levels of expression in the cells (data not shown).

Previous observations imply that HSPGs play a pivotal role in the apoE-mediated cholesterol efflux from macrophages. Lucas et al²¹ clarified that HSPGs sequester apoE molecules, leading to modulation in the synthesis and secretion of apoE from the cells. Obunike et al²² reported that the endogenously expressed apoE not only binds to cell surface HSPGs but also regulates the synthesis of HSPGs. Therefore, we explored the ability of apoE isoforms to modulate the efflux of cholesterol from macrophages with or without cell surface HSPGs.

When the production of cell surface HSPGs was inhibited, the endogenously expressed as well as exogenously derived apoE, regardless of its isoform, effectively reduced cholesterol in cholesterol-loaded RAW264.7 cells. There was no statistical difference among the exogenously derived 3 isoforms; however, a small but significant difference was observed among the 3 endogenously synthesized apoE isoforms. ApoE3 and E4 were more effective than apoE2. The probable explanation for this difference is the differential intracellular association of apoE isoforms to the ATP-binding cassette transporter, ABCA1. A recent report verified that ABCA1 plays an important role in cholesterol efflux mediated by endogenous apoE,²⁷ and our study clarified that ABCA1, which is induced by 8-Br-cAMP, is necessary for cholesterol efflux from RAW264.7 cells, not only by endogenous apoE but also by exogenous apoE. It is possible that endogenous apoE2 is least effective in ABCA1-mediated cholesterol efflux, leading to decreased efficiency in the efflux of cholesterol. This hypothesis needs to be tested with further experiments.

On the other hand, when the production of HSPGs was not inhibited, apoE2 reduced the cellular cholesterol content significantly, regardless of whether it was derived from outside or inside the cells. ApoE3 was less effective than apoE2 when synthesized in the cells; however, when exogenously derived, it reduced cellular cholesterol content significantly, which still showed a reduced tendency in cholesterol efflux compared with apoE2. ApoE4 had no significant effect on the cellular cholesterol content. The binding of apoE to cell surface HSPGs increased in the order E2<E3<E4. These observations, together with the findings obtained from the experiments without HSPGs, suggest that the secreted and exogenously derived apoE4 protein, as well as a portion of apoE3 protein, would be captured by the cell surface HSPGs and taken up and/or degraded by the cells. Support for this concept is the report by Lucas et al,²¹ which demonstrated that the apoE sequestered in the pericellular proteoglycan matrix is susceptible to cellular degradation. Ji et al²⁸ reported that the binding ability of ß-VLDL to HSPGs depends on the apoE isoforms in the order E4-ßVLDL<E3-ßVLDL=E2-ßVLDL; however, the binding of purified apoE to HSPGs was not different between apoE3 and apoE4. This finding is consistent with ours, considering that the lipoproteins examined in their study are ß-VLDL particles and those in ours are HDLs and lipid-poor apoEs. The size and composition of lipids of lipoprotein particles would modulate the presentation of apoE to HSPGs, thereby leading to the different binding ability to HSPGs among the different lipoprotein classes.

The LDL receptor is an another candidate that mediates the binding of apoE to the surface of macrophages²⁹ as well as HSPGs. The binding activity of apoE2 to the LDL receptor is <2% when compared with apoE3 and E4¹²; however, our data without HSPGs with exogenous apoE revealed that there is no difference in the efficiency of cholesterol efflux among the 3 apoE isoforms. This finding suggests that the LDL receptor is not a key player in the cholesterol efflux mediated by the cell surface-bound apoE or apoE-containing HDLs in macrophages.

The present study demonstrated that the ability of apoE to promote cholesterol efflux from macrophages is not fundamentally different among the apoE isoforms. However, in the physiological setting wherein HSPGs are produced, apoE2 is most and apoE4 least effective. Epidemiological studies have clarified that the incidence of coronary heart disease is generally lower in humans carrying at least 1 apoE2 allele than those possessing other apoE genotypes,³⁰ and the allele for apoE4 is an independent risk factor for coronary heart disease.^15,16 We speculate that under normolipidemic conditions, in which the insult by atherogenic lipoproteins is low, apoE2 functions most effectively for the reduction in cellular cholesterol among the 3 isoforms, thus protecting humans who possess apoE2 from atherosclerosis; on the other hand, apoE4 is least effective in the efflux and would lead to the increased atherosclerosis in humans who carry the apoE4 allele.

ApoE has been proposed to possess several biologic properties that could contribute to antiatherogenic effects.^31–34 The apoE polymorphism has been shown to affect some of these functions.³² In addition, apoE isoforms have been proved to possess differential direct cellular functions.³⁵ More investigations into the differential biologic effects of the isoforms would improve our understanding of the clinical observations that identified the differential effects of apoE isoforms on atherogenesis.^36,37

In summary, we demonstrated that the ability of apoE isoforms to promote cholesterol efflux from macrophages is fundamentally similar among the apoE isoforms, regardless of whether it is derived from outside the cell or synthesized inside the cell. Nevertheless, in the physiological setting where proteoglycans are produced by macrophages, the cell surface HSPGs would modulate the ability of apoE isoforms to promote cholesterol efflux, probably through the differential binding ability of each apoE isoform to HSPGs.

	Acknowledgments

 
This research was supported by grant-in-aid No. 10671058 from the Ministry of Education, Science and Culture of Japan. We are greatly indebted to Daniel J. Rader for critically reading the manuscript and helpful discussions.

Received August 12, 2002; accepted December 12, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Glass CK, Witztum JL. Atherosclerosis: the road ahead. Cell. 2001; 104: 503–516.

2. von Eckardstein A, Nofer JR, Assmann G. High density lipoproteins and arteriosclerosis: role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol. 2001; 21: 13–27.

3. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 1988; 240: 622–630.

4. Mazzone T. Apolipoprotein E secretion by macrophages: its potential physiological functions. Curr Opin Lipidol. 1996; 7: 303–307.

5. Beisiegel U. Receptors for triglyceride-rich lipoproteins and their role in lipoprotein metabolism. Curr Opin Lipidol. 1995; 6: 117–122.

6. Tsukamoto K, Tangirala R, Chun SH, Pure E, Rader DJ. Rapid regression of atherosclerosis induced by liver-directed gene transfer of apoE in apoE-deficient mice. Arterioscler Thromb Vasc Biol. 1999; 19: 2162–2170.

7. Tsukamoto K, Tangirala RK, Chun S, Usher D, Pure E, Rader DJ. Hepatic expression of apolipoprotein E inhibits progression of atherosclerosis without reducing cholesterol levels in LDL receptor-deficient mice. Mol Ther. 2000; 1: 189–194.

8. Bellosta S, Mahley RW, Sanan DA, Murata J, Newland DL, Taylor JM, Pitas RE. Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice. J Clin Invest. 1995; 96: 2170–2179.

9. Fazio S, Babaev VR, Murray AB, Hasty AH, Carter KJ, Gleaves LA, Atkinson JB, Linton MF. Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages. Proc Natl Acad Sci U S A. 1997; 94: 4647–4652.

10. Shimano H, Ohsuga J, Shimada M, Namba Y, Gotoda T, Harada K, Katsuki M, Yazaki Y, Yamada N. Inhibition of diet-induced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall. J Clin Invest. 1995; 95: 469–476.

11. Gregg RE, Zech LA, Schaefer EJ, Brewer HB Jr. Type III hyperlipoproteinemia: defective metabolism of an abnormal apolipoprotein E. Science. 1981; 211: 584–586.

12. Weisgraber KH, Innerarity TL, Mahley RW. Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site. J Biol Chem. 1982; 257: 2518–2521.

13. Gregg RE, Zech LA, Schaefer EJ, Stark D, Wilson D, Brewer HB Jr. Abnormal in vivo metabolism of apolipoprotein E4 in humans. J Clin Invest. 1986; 78: 815–821.

14. Tsukamoto K, Smith P, Glick JM, Rader DJ. Liver-directed gene transfer and prolonged expression of three major human apoE isoforms in apoE-deficient mice. J Clin Invest. 1997; 100: 107–114.

15. Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis. 1988; 8: 1–21.

16. Wilson PW, Myers RH, Larson MG, Ordovas JM, Wolf PA, Schaefer EJ. Apolipoprotein E alleles, dyslipidemia, and coronary heart disease: the Framingham Offspring Study. JAMA. 1994; 272: 1666–1671.

17. Cullen P, Cignarella A, Brennhausen B, Mohr S, Assmann G, von Eckardstein A. Phenotype-dependent differences in apolipoprotein E metabolism and in cholesterol homeostasis in human monocyte-derived macrophages. J Clin Invest. 1998; 101: 1670–1677.

18. Smith JD, Miyata M, Ginsberg M, Grigaux C, Shmookler E, Plump AS. Cyclic AMP induces apolipoprotein E binding activity and promotes cholesterol efflux from a macrophage cell line to apolipoprotein acceptors. J Biol Chem. 1996; 271: 30647–30655.

19. Carey DJ, Rafferty CM, Todd MS. Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells. J Cell Biol. 1987; 105: 1013–1021.

20. Shimada K, Ozawa T. Modulation of glycosaminoglycan production and antithrombin III binding by cultured aortic endothelial cells treated with 4-methylumbelliferyl-ß-D-xyloside. Arteriosclerosis. 1987; 7: 627–636.

21. Lucas M, Mazzone T. Cell surface proteoglycans modulate net synthesis and secretion of macrophage apolipoprotein E. J Biol Chem. 1996; 271: 13454–13460.

22. Obunike JC, Pillarisetti S, Paka L, Kako Y, Butteri MJ, Ho YY, Wagner WD, Yamada N, Mazzone T, Deckelbaum RJ, Goldberg IJ. The heparin-binding proteins apolipoprotein E and lipoprotein lipase enhance cellular proteoglycan production. Arterioscler Thromb Vasc Biol. 2000; 20: 111–118.

23. Lin CY, Huang ZH, Mazzone T. Interaction with proteoglycans enhances the sterol efflux produced by endogenous expression of macrophage apoE. J Lipid Res. 2001; 42: 1125–1133.

24. Murakami M, Horiuchi S, Takata K, Morino Y. Distinction in the mode of receptor-mediated endocytosis between high density lipoprotein and acetylated high density lipoprotein: evidence for high density lipoprotein receptor-mediated cholesterol transfer. J Biochem (Tokyo). 1987; 101: 729–741.

25. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrins vß3 and vß5 promote adenovirus internalization but not virus attachment. Cell. 1993; 73: 309–319.

26. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science. 1997; 275: 1320–1323.

27. Von Eckardstein A, Langer C, Engel T, Schaukal I, Cignarella A, Reinhardt J, Lorkowski S, Li Z, Zhou X, Cullen P, Assmann G. ATP binding cassette transporter ABCA1 modulates the secretion of apolipoprotein E from human monocyte-derived macrophages. FASEB J. 2001; 15: 1555–1561.

28. Ji ZS, Pitas RE, Mahley RW. Differential cellular accumulation/retention of apolipoprotein E mediated by cell surface heparan sulfate proteoglycans: apolipoproteins E3 and E2 greater than E4. J Biol Chem. 1998; 273: 13452–13460.

29. Zhao Y, Mazzone T. LDL receptor binds newly synthesized apoE in macrophages: a precursor pool for apoE secretion. J Lipid Res. 1999; 40: 1029–1035.

30. Mahley RW, Huang Y, Rall SC Jr. Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia): questions, quandaries, and paradoxes. J Lipid Res. 1999; 40: 1933–1949.

31. Ishigami M, Swertfeger DK, Granholm NA, Hui DY. Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase. J Biol Chem. 1998; 273: 20156–20161.

32. Miyata M, Smith JD. Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by oxidative insults and ß-amyloid peptides. Nat Genet. 1996; 14: 55–61.

33. Riddell DR, Graham A, Owen JS. Apolipoprotein E inhibits platelet aggregation through the L-arginine: nitric oxide pathway: implications for vascular disease. J Biol Chem. 1997; 272: 89–95.

34. Kelly ME, Clay MA, Mistry MJ, Hsieh-Li HM, Harmony JA. Apolipoprotein E inhibition of proliferation of mitogen-activated T lymphocytes: production of interleukin 2 with reduced biological activity. Cell Immunol. 1994; 159: 124–139.

35. Nathan BP, Bellosta S, Sanan DA, Weisgraber KH, Mahley RW, Pitas RE. Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro. Science. 1994; 264: 850–852.

36. van Bockxmeer FM, Mamotte CD, Gibbons FR, Taylor RR. Apolipoprotein 4 homozygosity: a determinant of restenosis after coronary angioplasty. Atherosclerosis. 1994; 110: 195–202.

37. van Bockxmeer FM, Mamotte CD, Gibbons FA, Burke V, Taylor RR. Angiotensin-converting enzyme and apolipoprotein E genotypes and restenosis after coronary angioplasty. Circulation. 1995; 92: 2066–2071.