Introduction

The strong inverse correlation between high-density lipoprotein (HDL) plasma levels and coronary heart disease has long been recognized in epidemiological studies^1,2. Apolipoprotein A-I (apoA-I), the major protein component of HDL, is essential for the correct assembly and stability of HDL^3,4,5. ApoA-I is also critically involved in mediating the interaction of HDL with peripheral and hepatic cells to facilitate the removal of excess cellular cholesterol and return to the liver in a process referred to as reverse cholesterol transport (RCT)^6,7. The initial step in the process of RCT is the interaction of small, lipid-poor HDL with cell surfaces to activate cholesterol efflux. In fact, lipid-free apoA-I is more efficient than plasma HDL at promoting cholesterol efflux from macrophage-derived foam cells⁸.

ApoA-I accepts cholesterol translocated by the ATP binding cassette (ABC) transporters. The ABC superfamily comprises a large number of membrane proteins, which transport a variety of substrates across cell membranes⁹. Several members of this gene family are involved in transporting cholesterol and phospholipid into or out of the cell. ABCA1 is thought to be critical in apoA-I-mediated cholesterol and phospholipid efflux and to play an important role in HDL maturation and RCT^{10,11,12,13,14,15,16,17,18,19}. Recently, the genetic cause of Tangier disease, a low-HDL syndrome with accelerated atherosclerosis, was positively attributed to mutations in the ABCA1 gene^20,21. ABCG1 is another member of the ABC family with a high sequence and expression pattern similarity to ABCA1²². Up-regulation of ABCG1 was observed exclusively during monocyte differentiation into macrophages and foam cell formation after acetylated LDL (acLDL) loading^23,24. Overexpression of ABCG1 has also been found in macrophages from patients with Tangier disease²⁵.

Considerable evidence indicates that overexpression of human apoA-I reduces atherosclerosis in animal models^26,27,28,29. However, it is not known whether this protective effect is due to an increase in plasma HDL concentration or to the presence of apoA-I within the atherosclerotic lesions, as a stimulus to cholesterol efflux from arterial macrophages.

Macrophages are involved in all stages of atherogenesis, from early fatty streak formation to the development of complicated lesions leading to myocardial infarction^30,31. Therefore, therapeutic interventions that act to promote cholesterol efflux and modulate the inflammatory response in macrophages might interrupt lesion formation and protect against atherogenesis. ApoA-I is synthesized by the liver and small intestine but, unlike apoE, is not naturally synthesized by macrophages^32,33,34. Recently, we have demonstrated that expression of human apoA-I in macrophages increases cholesterol efflux and protects apoA-I-deficient mice against high-fat diet-induced atherosclerosis^35,36. Because apoA-I-deficient mice have low cholesterol and develop only small lesions in the aortic root, the relevance of these observations to conditions of truly accelerated atherosclerosis could not be established. The current study was performed to test the hypothesis that expression of human apoA-I in bone marrow-derived macrophages produces beneficial effects on the arterial wall in apoE-deficient mice with severe hypercholesterolemia and severe atherosclerotic lesions. We observed a significant reduction in atherosclerotic lesions in mice that received apoA-I-expressing bone marrow, accompanied by increased ABCA1and ABCG1 mRNA levels in peritoneal macrophages isolated from these mice. Our results support the view that stem cell-based gene delivery of apoA-I represents a viable tool for the molecular therapy of atherosclerosis.

Results

Reconstitution of ApoE^-/- recipient mice with transduced marrow

We transplanted 12-week-old female ApoE^-/- mice with bone marrow cells transduced with MFG parental virus (MFG) or MFG expressing human apoA-I (MFG-HAI). PCR analysis of bone marrow isolated from recipient mice 16 weeks after transplantation confirmed the presence of the human apoA-I sequence (Fig. 1A). Also, we confirmed the presence of human apoA-I in the culture medium of apoE^-/-MFG-HAI peritoneal macrophages by Western blot analysis (Fig. 1B). We detected no human apoA-I in the culture medium from control apoE^-/-MFG peritoneal macrophages.

Figure 1.

Detection of human apoA-I in bone marrow and peritoneal macrophages from recipient mice. (A) PCR genotyping for human apoA-I in bone marrow cells from ApoE^-/- recipient mice 16 weeks post-BMT. Lane 1, 100 bp DNA ladder; lanes 2 and 13, blank; lane 3, negative control; lanes 4 through 6, samples from mice reconstituted with apoE^-/-MFG; lanes 7 through 12, samples from mice reconstituted with apoE^-/-MFG-HAI; lane 14, positive control. (B) Detection of human apoA-I secreted into culture medium of peritoneal macrophages. Lanes 1 and 2, 10 l of human serum; lane 3, unconditioned DMEM; lanes 4, culture medium from apoE^-/-MFG; lanes 5 and 6, culture medium from apoE^-/-MFG-HAI macrophages.

Full figure and legend (178K)

Human apoA-I levels in the serum of apoE^-/-MFG-HAI mice peaked at 3 weeks post-BMT (351 11 ng/dl) by enzyme-linked immunosorbent assay (ELISA) and decreased by 40% (148 65 ng/dl) by 16 weeks post-BMT (Fig. 2A). We detected no human apoA-I in the serum of apoE^-/-MFG-HAI mice before BMT or in recipient mice reconstituted with apoE^-/-MFG marrow before or after transplantation.

Figure 2.

Human and mouse apoA-I levels in serum of recipient mice. (A) Human apoA-I levels in serum measured by ELISA. Closed squares represent samples from mice reconstituted with apoE^-/-MFG marrow. Closed circles are samples from recipients of apoE^-/-MFG-HAI marrow. (B) Endogenous mouse apoA-I in serum. Lane 1, ApoE^-/- serum; lane 2, blank; lanes 3 through 7, samples taken from mice before transplantation and at 3, 8, 12, and 16 weeks post-BMT.

Full figure and legend (155K)

Because high levels of exogenous human apoA-I have been shown to suppress the production of endogenous mouse apoA-I^29,37, we performed Western blotting analysis to determine whether small amounts of human apoA-I produced by macrophages in the apoE^-/-MFG-HAI mice would affect endogenous circulating levels of mouse apoA-I protein. We detected no changes in serum mouse apoA-I level in mice after transplantation of apoE^-/-MFG and apoE^-/-MFG-HAI during the course of the study (Fig. 2B).

Effects of macrophage expression of human ApoA-I on the lipid profile

We observed no significant differences in serum cholesterol and triglyceride between recipients reconstituted with apoE^-/-MFG or apoE^-/-MFG-HAI at 16 weeks post-BMT (Table 1). We found no differences in baseline serum cholesterol levels between groups; however, there was a slight elevation in the baseline serum triglyceride levels in the apoE^-/-MFG-HAI group.

Table 1 - Total serum cholesterol and triglyceride levels in recipient mice before and after bone marrow transplantation.

Full table

Examination of the distribution of cholesterol at 16 weeks post-BMT revealed similar lipoprotein profiles between the groups. Interestingly, HDL cholesterol levels were slightly increased in the serum of apoE^-/-MFG-HAI mice compared to apoE^-/-MFG mice (Fig. 3) at 16 weeks post-BMT. HDL cholesterol was 29 1 mg/dl (n = 3) in the apoE^-/-MFG-HAI mice compared to the baseline value of 22 1 mg/dl (n = 3). Transplantation alone did not affect HDL levels in apoE^-/-MFG mice (19 1 mg/dl before BMT and 20 1 mg/dl at 16 weeks post-BMT). The distribution of human apoA-I in serum of apoE^-/-MFG-HAI mice was mainly within the HDL fraction (>80%). No human apoA-I was detected in the VLDL fraction (data not shown).

Figure 3.

Effect of human APOAI gene transduction on distribution of serum lipoprotein cholesterol in recipient mice. Distribution of serum lipoprotein cholesterol at 16 weeks post-BMT was determined by FPLC followed by cholesterol analysis of each fraction. Each data point represents three pooled samples. Fractions 15 through 20 contain VLDL, fractions 21 through 28 contain LDL, fractions 29 through 34 contain HDL. Fractions 35 through 40 are the non-lipoprotein-associated proteins.

Full figure and legend (71K)

Effects of human ApoA-I produced by macrophages on atherosclerosis

The mean lesion area at 8 weeks post-BMT was 30% lower in the apoE^-/-MFG-HAI mice (93,659 8716 m², n = 18) than in the apoE^-/-MFG mice (132,980 13,871 m², n = 14; p < 0.05, Fig. 4). This effect was sustained throughout the course of the study, and the mean lesion area at 16 weeks post-BMT was also 30% lower in the apoE^-/-MFG-HAI mice (256,934 28,219 m², n = 14) compared to apoE^-/-MFG mice (372,886 24,862 m², n = 9; p < 0.01). Analyses of the atherosclerotic lesions by immunohistochemistry staining using a macrophage-specific antibody, MOMA-2, showed that the ratios of macrophage-positive area to lipid-content area stained by Oil-Red-O were similar in both apoE^-/-MFG-HAI (36 3%, n = 12) and apoE^-/-MFG (33 4%, n = 9) mice.

Figure 4.

Quantification of atherosclerotic lesion area in recipient reconstituted with donor marrow. Data are expressed as mean lesion area per section in square micrometers. Error bars represent the SEM. ^*p < 0.05 compared with apoE^-/-MFG at 8 weeks post-BMT. ^**p < 0.01 compared with apoE^-/-MFG at 16 weeks post-BMT.

Full figure and legend (92K)

Effect of macrophage expression of human ApoA-I on LCAT activity

To determine the level of cholesterol esterification in mice reconstituted with apoE^-/-MFG-HAI marrow, we analyzed serum LCAT activity. There was no difference in LCAT activity in serum between apoE^-/-MFG (1.24 0.03, n = 8) and apoE^-/-MFG-HAI mice (1.30 0.08, n = 10) before BMT. The apoE^-/-MFG-HAI mice had a significant increase (10%) in LCAT activity (1.46 0.03) at 16 weeks post-BMT compared to the apoE^-/-MFG mice (1.29 0.04; p < 0.01).

Expression of human ApoA-I in ApoE^-/- macrophages and up-regulation of ABCA1 and ABCG1

The expression of ABCA1and ABCG1 mRNA in the apoE^-/-MFG-HAI peritoneal macrophages (n = 8) was 2.1- and 3.7-fold higher, respectively, than that in the apoE^-/-MFG macrophages (n = 3, Fig. 5A) as measured by real-time quantitative RT-PCR. We observed a further increase to 3.9- and 5.2-fold, ABCA1 and ABCG1 mRNA levels, respectively, upon acLDL treatment (Fig. 5B).

Figure 5.

Quantification of ABCA1and ABCG1mRNA in peritoneal macrophages. Quantification of ABC transporter mRNA was performed using real-time quantitative RT-PCR. The quantity of ABCA1or ABCG1mRNA was expressed relative to the expression levels in apoE^-/-MFG macrophages. (A) Relative quantity of ABCA1 mRNA and (B) ABCG1 mRNA. Error bars represent the SE. ^*p < 0.05, ^**p < 0.02 compared with apoE^-/-MFG.

Full figure and legend (105K)

To determine whether the apoA-I secreted by the macrophage affects cholesterol efflux similar to extracellular apoA-I, we incubated ApoE^-/- peritoneal macrophages with exogenous apoA-I at concentrations (0.5 to 2.0 g/ml) close to those produced by the transgenic apoA-I-expressing macrophages. This resulted in a dose-dependent and significant increase in ABCA1-mediated cholesterol efflux from ApoE^-/- cells. In addition, the relative quantity of ABCA1 and ABCG1 mRNA was increased more than 3-fold upon loading with acLDL (Figs. 6A and 6B). Interestingly, the transgenic expression of human apoA-I by ApoE^-/- macrophages had similar effects on cholesterol efflux compared to those produced by the addition of exogenous apoA-I to ApoE^-/- macrophages.

Figure 6.

Effects of apoA-I on cholesterol efflux in ApoE^-/- macrophages. ApoE^-/- macrophages (solid bars) or ApoE^-/- macrophages expressing human apoA-I (open bars) were pretreated with exogenous apoA-I and then used for cholesterol efflux and quantification of ABCA1 and ABCG1mRNA. (A) ABCA1-mediated efflux of [³H]cholesterol in the presence of apoA-I (15 g/ml for 4.5 h). Values are means SD (n = 8 in each group). Comparisons are made with ApoE^-/- macrophages in the absence of apoA-I. ^*p < 0.001. (B and C) Relative quantity of ABCA1 and ABCG1 mRNA levels, respectively, in ApoE^-/- macrophages or ApoE^-/- macrophages expressing endogenous apoA-I, with or without exogenous apoA-I. Values represent pooled samples of four. (D) Western blotting analysis of ABCA1 in ApoE^-/- macrophages or ApoE^-/- macrophages expressing endogenous apoA-I. 30 g of cell lysate protein was loaded in each lane without cAMP treatment and 20 g of protein was loaded in the two lanes with cAMP treatment.

Full figure and legend (139K)

Western analysis showed that the apoA-I-expressing ApoE^-/- macrophages have higher basal level ABCA1 protein expression than ApoE^-/- macrophages. Either endogenous or exogenous expression of human apoA-I increases the amount of ABCA1 protein. These effects were further induced by acLDL treatment (Fig. 6D).

Discussion

In the present study, a retroviral transduction approach was used to determine whether human apoA-I produced by macrophages would protect ApoE^-/- mice with preexisting plaques against the progression of atherosclerotic lesions. Reconstitution of ApoE^-/- recipient mice with apoE^-/-MFG-HAI significantly reduced (30%) the mean lesion area compared to that of apoE^-/-MFG mice at both 8 and 16 weeks post-BMT. This difference in the atherosclerosis was accompanied by a slight increase in serum HDL level, possibly a consequence of increased LCAT activity. However, there was no significant increase in either human or mouse apoA-I in the plasma. Therefore, the delayed progression of preexisting atherosclerotic lesions was most likely due to the local expression of human apoA-I in macrophages located in the vascular wall.

Excess accumulation of cholesterol in the arterial wall is a hallmark of atherosclerosis. Substantial efforts have been made to promote cholesterol efflux and reverse cholesterol transport to prevent or treat atherosclerosis³¹. Rubin and others have shown that overexpression of a human APOAI transgene from the liver, causing a twofold increase in HDL cholesterol and apoA-I levels, leads to complete protection from the development of atherosclerosis in C57BL/6²⁷ and hyperlipidemic mice^28,29. Moreover, inhibition of atherosclerosis was found in cholesterol-fed human APOAI transgenic rabbits. Cholesterol efflux is increased when Fu5HA hepatoma cells are incubated with serum from APOAI transgenic rabbits compared with those incubated with serum from control rabbits²⁶. A significant reduction of atherosclerosis was also found in somatic gene transfer of human APOAI into LDL receptor-deficient mice fed with a Western-type diet^38,39. We have recently shown that macrophages expressing human apoA-I increase cholesterol efflux by 40% compared to control macrophages³⁶ and reduce diet-induced plaque size in apoA-I-deficient mice³⁵. Collectively, these studies suggest that human apoA-I increases cholesterol efflux and protects against atherosclerosis development in animal models.

Cholesterol efflux can occur by both aqueous diffusion⁴⁰ and energy-dependent mechanisms, including those mediated by ABC transporters that result in the transfer of cellular cholesterol to lipid-poor apolipoproteins^19,41. Recent studies have reported that increased ABCA1 expression results in increased efflux of cellular cholesterol and phospholipids to exogenous lipid-free apolipoproteins such as apoA-I^{15,42,43,44,45}. The enhanced lipid efflux is associated with increased apoA-I binding to ABCA1 on the cell surface⁴⁶.

We have observed a significant increase in ABCA1 and ABCG1 mRNA in transduced ApoE^-/- macrophages expressing human apoA-I upon acLDL treatment compared to the control ApoE^-/- macrophages. The exact mechanisms by which apoA-I up-regulates the expression of ABCA1 and ABCG1 at the transcriptional level are not known. Because the endogenous apoA-I affects both ABCA1 and ABCG1 mRNA and was evident only upon cholesterol loading, we propose that apoA-I may influence cholesterol trafficking and regulate the expression of ABC transporters through an LXR-mediated pathway.

Recently, Wang et al.⁴⁷ reported that apoA-I binds directly to ABCA1 and inhibits calpain protease-mediated degradation of ABCA1 on the cell membrane, therefore leading to an increase in ABCA1 protein mass. Arakawa⁴⁸ also showed that lipid-free apoA-I stabilizes ABCA1 by protecting it from protease-mediated degradation. Downstream events would include activation of cholesterol efflux, intracellular cholesterol depletion, and compensatory reduction in ABCA1 mRNA levels. However, the links between ABCA1 message and protein levels vis-a-vis the many separate circumstances of altered cholesterol homeostasis are not as firmly defined.

For example, Huang et al. reported that macrophage cholesterol efflux mediated by endogenous apoE expression is independent of ABCA1. Higher ABCA1 protein levels were found in J774 murine macrophages (which do not express apoE) than in apoE-transfected J774 cells⁴⁹. Similarly, we have observed higher level of ABCA1 and ABCG1 mRNA in peritoneal macrophages isolated from ApoE^-/- mice compared to C57BL/6 mice (Y.R. Su and S. Fazio, unpublished observation). This is not in line with the expected stabilization of ABCA1 from the amphipathic helices of apoE. Moreover, Denis et al.⁵⁰ have shown that sterols stimulate ABCA1 transcription in monocyte-derived macrophages, whereas Maor et al.⁵¹ demonstrated that cellular oxysterols were seven times higher in monocytes derived from apoE-deficient mice in comparison to monocytes from control C57BL/6 mice. The total oxysterols were even higher (16 times over control) in macrophages isolated from aortas of the atherosclerotic apoE-deficient mice. The increase in cellular oxysterols in the apoE-deficient macrophages could up-regulate ABCA1 and ABCG1 message through the LXR-mediated pathway. Our results suggest that macrophage expression of apoA-I can compensate in part for apoE deficiency. However, since the apoE-dependent efflux pathway remains defective, the net cholesterol efflux from apoA-I-producing macrophages in the artery wall may still not be fully restored.

In contrast to ABCA1, little information is available on the function of ABCG1. In recent reports, ABCG1 appears to be regulated by cholesterol loading in macrophages and is expressed by macrophage-derived foam cells in human atherosclerotic plaques^23,25. In the apoE^-/- MFG-HAI macrophages, ABCG1 gene expression was also up-regulated under acLDL loading compared with the apoE^-/-MFG macrophages. These data indicate that not only ABCA1 but also ABCG1 might play an important role in macrophage cholesterol homeostasis.

Our in vitro experiments in ApoE^-/- macrophages loaded with acLDL showed that endogenous human apoA-I has an effect similar to that of exogenous apoA-I on stimulating cholesterol efflux. These results suggest that the local effects of human apoA-I produced by macrophages can promote removal of excess cholesterol in the plaque. In normolipidemic human plasma, lipid-free apoA-I levels are around 5–10% of the total plasma apoA-I⁵². The lipid-free apoA-I particles are able to enter the subendothelial space and promote the removal of excess cholesterol from peripheral cells. If the lipid-free human apoA-I produced by macrophages in the subendothelial space can effectively promote cholesterol efflux, high levels of apoA-I in plasma may not represent a necessary condition to obtain beneficial effects on the vascular wall. Modulating atherogenesis by increasing the apoA-I concentration locally in the vascular wall through a gene-therapy approach may represent a viable therapeutic avenue alternative to that aimed at increasing HDL.

Other mechanisms could also be involved in apoA-I-mediated reduction of atherosclerosis. We observed a slight increase in LCAT activity that was associated with increased HDL cholesterol levels in apoE^-/-MFG-HAI mice. In addition, apoA-I has been shown to have anti-inflammatory effect on vascular wall endothelial cells. Infusion of apoA-I prevents the oxidation of low-density lipoprotein by the artery wall cells^53,54. ApoA-I inhibits the production of interleukin-1 and tumor necrosis factor- from monocytes, therefore it reduces the proinflammatory factors for atherosclerosis⁵⁵.

Understanding the molecular causes of complex cardiovascular diseases such as atherosclerosis will lead to the identification of new targets for gene therapy. Hematopoietic stem cells (HSC) are ideal tools for the development of gene therapy modalities of several diseases^56,57. The development of new vectors that are more effective for gene transfer to HSC with a long-term expression, such as lentivirus and adeno-associated virus, may provide more promising approaches to cardiovascular gene therapy targeted to HSC. Our data support the view that expression of antiatherogenic apolipoproteins, such as human apoA-I, from macrophages may translate into a viable gene therapy approach to atherosclerosis.

Materials and methods

Animal procedures

ApoE^-/- mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME). All mice used in this study were maintained in microisolator cages on an autoclaved rodent chow diet (PMI Feeds, Inc., St. Louis, MO) containing 4.5% fat. Mice were healthy and there were no differences in feeding pattern or body weight between groups throughout the study. Animal care and experimental procedures were performed according to the regulations of Vanderbilt University's Institutional Animal Care and Usage Committee.

Construction of an MFG retroviral vector containing human apoA-I cDNA

The human apoA-I cDNA was cloned into the MFG retroviral vector and the PT67 packaging cell line was transfected with MFG or MFG-containing MFG-HAI as previously described³⁵.

Mice were matched for age and serum cholesterol levels before assignment to experimental or control groups. At 12 weeks of age, one group of nontransplanted mice (defined as a baseline control) was euthanized for quantification of atherosclerotic lesions. The remaining mice were divided into two groups, lethally irradiated (9 Gy), and transplanted with apoE^-/- MFG (control group) or apoE^-/- MFG-HAI marrow. Mice were euthanized at 8 and 16 weeks post-BMT for evaluation.

Bone marrow transplantation

A retroviral transduction approach was performed as previously described³⁵. Briefly, bone marrow cells were collected and precultured by placing 5.0 10⁶ cells in 10 ml of Marrow MAX (Invitrogen) medium supplemented with FBS, human stroma cell conditioned medium, and L-glutamine (Invitrogen) for 48 h. The remaining bone marrow manipulations were done as previously described^58,59. Lethally irradiated 12-week-old female ApoE^-/- recipient mice were given 4.0–8.0 10⁶ retroviral-transduced bone marrow cells/mouse in 0.2 ml RPMI 1640 medium intravenously.

Polymerase chain reaction for genotyping

DNA samples were extracted from recipient bone marrow cells at 16 weeks post-BMT. PCR was performed as previously described³⁶.

Primary culture of peritoneal macrophage

Cells were collected 3 days after intraperitoneal injection of 3% thioglycollate as described⁶⁰. Peritoneal cells were collected and plated onto six-well plates in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. After 24 h, culture media were changed to fresh DMEM without FBS and collected from each well at 72-h intervals for Western blot analysis. For quantitative analysis of ABCA1 and ABCG1 mRNA in peritoneal macrophages, cells were placed in DMEM with 4% FBS, with or without 70 g/ml acLDL for 24 h.

Western blotting analysis for human apoA-I and mouse apoA-I

To detect human apoA-I secreted into culture media, 1.0 ml of peritoneal macrophage culture medium was concentrated to 10 l with Microcon YM-10 (Millipore Corp., Waltham, MA). Concentrated culture media were separated on a 10% NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA) and subsequently transferred to the nitrocellulose membrane. The membranes were incubated with a polyclonal goat anti-human apoA-I antibody (BioDesign International, Saco, ME) or rabbit anti-mouse apoA-I antibody (BioDesign International) and a horseradish peroxidase-conjugated anti-goat IgG (Sigma) as a secondary antibody. Specific bands were visualized by chemiluminescence with the ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).

ELISA for detection of human apoA-I

Serum levels of human apoA-I-transduced bone marrow were determined by a specific ELISA system as previously described³⁶.

Lipid and lipoprotein analysis

Fasting blood samples were collected from retro-orbital venous plexus puncture using heparinized tubes (Medeva Pharmaceuticals, Inc., Rochester, NY). Total cholesterol and triglyceride levels were measured by enzymatic methods as previously described^35,61.

Serum samples from recipient mice were subjected to fast-performance liquid chromatography (FPLC) as previously described⁶¹. The distribution of serum lipoprotein cholesterol represents a pool of three individual samples from each group.

Assessment of atherosclerotic lesions

For quantification of proximal arterial lesions, at baseline (12-week-old) and at 8 or 16 weeks post-BMT, mice were euthanized and the proximal aorta was isolated to evaluate atherosclerotic lesion area according to the technique of Paigen et al.⁶², adapted for computer analysis⁶¹. The operators were blinded to the group assignment of the sections analyzed. The ratio of macrophage/lipid staining in the arterial lesion was examined by immunohistochemistry as described³⁵. The percentage of macrophages in the lesion was calculated as a ratio of the macrophage-stained area to the Oil-Red-O-stained area.

LCAT activity assay

The LCAT activity was measured by following the conversion of cholesterol to cholesterol ester with an LCAT assay kit (Roar Biomedical, Inc., New York, NY) according to the manufacturer's protocol. In brief, 2 l of serum was incubated with the fluorescently labeled cholesterol for 60 min at 37°C, and the conversion of cholesterol (470 nm) to cholesterol ester (390 nm) at 340 nm excitation was determined by fluorescence microplate reader (SPECTRAmax GEMINI; Molecular Devices Co., Sunnyvale, CA). The LCAT activity was expressed as a ratio of the two emission intensities (390/470).

Real-time quantitative RT-PCR for ABCA1 and ABCG1

Total RNA was isolated from peritoneal macrophages at 16 weeks post-BMT using the Trizol reagent (Invitrogen) according to the manufacturer's instruction. Relative quantification of ABCA1 and ABCG1 mRNAs was performed using a FAM-labeled Taqman probe with the TaqMan One-Step RT-PCR Master Mix reagent kit (Applied Biosystems, ABI, Foster City, CA, P/N 4309169) on an ABI Prism 7700 sequence detection system (ABI) according to the method developed by Su et al.⁶³. The relative quantities of ABCA1 and ABCG1 message were normalized with 18S ribosomal RNA as an internal control. ABCA1 or ABCG1 mRNA expression levels in different macrophage types were expressed relative to the expression in apoE^-/-MFG macrophages. The data were analyzed using the comparative C_T method and were conformed by the standard curve method.

Western blotting analysis of murine ABCA1

Cellular extracts from cultured peritoneal macrophages were separated by 3–8% NuPAGE Tris-acetate gels (Novex, San Diego, CA) and transferred to nitrocellulose membrane. Murine ABCA1 was detected with a primary antibody (Novus Biological, Littleton, CO) and visualized by a chemiluminescent ECL Plus (Amersham Pharmacia Biotech) according to Bortnick et al.⁴³.

Lipoprotein isolation and modification

LDL (d = 1.019 to 1.063 g/ml) was isolated from human plasma by sequential ultracentrifugation. AcLDL was prepared by repeated addition of acetic anhydride (Sigma) to LDL in a sodium acetate solution and dialyzed in 0.15 M NaCl and 0.3 mM EDTA at 4°C for 2 days⁶⁴.

[³h]Cholesterol efflux studies with macrophages

Cholesterol efflux using macrophages isolated from ApoE^-/- mice or ApoE^-/- expressing the human APOAI transgene was measured by a modified procedure from Lin et al.⁶⁵. Peritoneal macrophages were elicited with thioglycollate (3%) and plated at a density of 4 10⁵ cells/well on 24-well plates in DMEM/10% FBS. Cells were cultured in DMEM/RPMI (1/1) with 1% FBS and 1% Nutridoma (Roche) for 24 h prior to experiments. Cells were then incubated with 70 g/ml acLDL and 1.5 Ci/ml [³H]cholesterol (NEN) in serum-free DMEM/RPMI (1/1) with 1% Nutridoma for 24 h. During this loading period, macrophages were treated with 0, 0.5, or 2 g/ml exogenous human apoA-I (Calbiochem) followed by rinsing in DMEM/RPMI (1/1) with 1% Nutridoma to remove the acLDL and apoA-I. After rinsing, 15 g/ml human apoA-I was added to measure cholesterol efflux mediated by the ABCA1 transporter. Media were collected after 4.5 h, centrifuged, and counted by scintillation. Cells were lysed with 0.1 N NaOH and counted by scintillation. Cholesterol efflux was calculated from the counts in media and expressed as a percentage of the total counts (medium and lysate).

Statistical analysis

Statistical analysis was performed with GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). Results were expressed as the means SE. Intragroup and between-group comparisons were achieved using either one-way ANOVA followed by the Bonferroni's posttest when statistical significance was detected or the unpaired two-tailed t test when appropriate.

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Acknowledgements

This work was supported in part by National Institutes of Health Grants HL65709 and HL57986 to Sergio Fazio and HL53989 to MacRae F. Linton. Sergio Fazio and MacRae F. Linton were supported by Established Investigatorships of the American Heart Association. Vlademir Babaev is supported by American Heart Association Grant SE0160160B. Alyssa H. Hasty was supported by Mechanisms in Cardiovascular Research Training Grant T32HC07411. Amy S. Major is the recipient of an NRSA (HL10206-03). This study was also supported by the Lipid, Lipoprotein and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Physiology Center (NIH DK59637-01). We thank Tianli Zhu, Janet Matthews, and Youmin Zhang for technical assistance.