Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARα, β/δ, and

Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARα, β/δ, and

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  Research article 1564  The Journal of Clinical Investigation http://www.jci.org    Volume 114   Number 11   December 2004 Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPAR α , β  /  δ , and γ  Andrew C. Li, 1  Christoph J. Binder, 2  Alejandra Gutierrez, 3  Kathleen K. Brown, 4  Christine R. Plotkin, 5  Jennifer W. Pattison, 2  Annabel F. Valledor, 1  Roger A. Davis, 3  Timothy M. Willson, 6  Joseph L. Witztum, 2  Wulf Palinski, 2  and Christopher K. Glass 1,2 1 Department of Cellular and Molecular Medicine and 2 Department of Medicine, University of California, San Diego, La Jolla, California, USA. 3 Department of Biology, San Diego State University, San Diego, California, USA. 4 Department of Metabolic Diseases, GlaxoSmithKline, Research Triangle Park, North Carolina, USA. 5 Genomics Core, Center for AIDS Research, Veterans Medical Research Foundation, La Jolla, California, USA. 6 Department of Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, North Carolina, USA. PPAR  α , β / δ , and γ  regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor–ABCA1 (LXR-ABCA1) pathway. To investigate the potential impor-tance of these activities in vivo, we performed a systematic analysis of the effects of PPAR  α , β , and γ  agonists on foam-cell formation and atherosclerosis in male LDL receptor–deficient (LDLR  –/– ) mice. Like the PPAR  γ  agonist, a PPAR  α -specific agonist strongly inhibited atherosclerosis, whereas a PPAR  β -specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPAR  α  and PPAR  γ  agonists, but not the PPAR  β  agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpect-edly, PPAR  α  and PPAR  γ  agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPAR  α  required LXRs, activation of PPAR  γ  reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmaco-logically to inhibit the development of atherosclerosis. Introduction PPAR  α , PPAR  β  (also referred to as δ ), and PPAR  γ  are members of the nuclear receptor superfamily that regulate gene expression in response to the binding of fatty acids and their metabolites (reviewed in refs. 1–4). PPARs regulate the expression of genes that control lipid metabolism by binding as heterodimers with retinoid X receptors to PPAR response elements in the promoter and/or enhancer regions of these genes (1, 5). PPARs also inhibit expression of proinflammatory genes in a ligand-dependent manner, in part by inhibiting the actions of NF- κ B and activator protein 1 (AP-1) family members (6–10). Because of their ability to regulate genes involved in both lipid homeostasis and inflammation, PPARs are promising targets for the development of novel antiatherogenic treatments. In addition to influencing global aspects of lipid and glucose metabolism, PPARs are expressed in the major cell types that make up atherosclerotic lesions, including macrophages, smooth muscle cells, lymphocytes, and endothelial cells, suggest-ing that ligands for these receptors may act both systemically and locally to influence lesion development (8, 11–15).In vitro studies indicate that PPARs can exert both atherogenic and antiatherogenic effects on gene expression. Potential atherogenic effects include the ability of PPAR  α  agonists to stimulate the production of monocyte chemoattractant protein 1 (MCP-1) in endothelial cells (16), which would be expected to enhance recruitment of monocytes into lesions (17–19). PPAR  γ  agonists stimulate expression of the scavenger receptor CD36 in macrophages, which facilitates uptake of oxidized LDL (oxLDL) and contributes to the development of atherosclerosis in mouse models (20, 21). Potential antiatherogenic consequences of acti- vating PPARs include the ability of PPAR  α  and PPAR  γ  agonists to inhibit expression of inflammatory response genes — including IFN- γ , IL-1 β , IL-6, TNF- α , and C-C chemokine receptor 2 — that promote the recruitment of monocytes and T cells into lesions and their subsequent differentiation and activation (8, 22–25). PPAR  α  and PPAR  γ  have also been demonstrated to stimulate cholesterol efflux in cultured macrophages by inducing the expression of liver X receptor α  (LXR  α ), which in turn activates expression of ABCA1 and other genes involved in cholesterol efflux (26, 27). PPAR  β  agonists exert variable effects on cholesterol efflux. A PPAR  β -specific agonist (GW501516) has been shown to enhance reverse cholesterol transport in a human macrophage cell line (THP-1), in skin fibroblasts (1BR3N), and in intestinal cells (FHS74), and to increase plasma HDL levels in obese, insulin-resistant rhesus monkeys (28). On the other hand, a different PPAR  β  agonist has Nonstandard abbreviations used:  ACAT, Acyl-CoA: cholesterol acyltransferase; acLDL, acetylated LDL; agLDL, aggregated LDL; AP-1, activator protein 1; Bcl6, B cell leukemia/lymphoma 6; DKO, double knockout; DTA, descending thoracic aorta; EC 50 , median effective concentration; FPLC, fast-performance liquid chro-matography; HC, high cholesterol; LDLR  –/– , LDL receptor–deficient; LXR, liver X receptor; MCP-1, monocyte chemoattractant protein 1; oxLDL, oxidized LDL;  Rag-1 , recombinase   activating gene 1 ; SRA, scavenger receptor A. Conflict of interest:  Timothy M. Willson and Kathleen K. Brown are employees of GlaxoSmithKline. Citation for this article:    J. Clin. Invest.   114 :1564–1576 (2004). doi:10.1172/JCI200418730.  Related Commentary, page 1538  research article  The Journal of Clinical Investigation   http://www.jci.org Volume 114 Number 11 December 2004 1565 also been shown to promote lipid accumulation in THP-1 cells and primary human macrophages (29).Studies of PPAR  α  in mouse models of atherosclerosis have yielded conflicting results. Mice lacking both PPAR  α  and apoE developed less atherosclerosis than mice lacking only apoE when fed a high-fat, high-cholesterol (HC) diet, suggesting a net atherogenic effect of PPAR  α  in this model (30). In contrast, the PPAR  α  agonist fenofibrate exerted minimal antiatherogenic effects in apoE-deficient mice (31, 32), but a more pronounced effect in apoE-deficient mice carrying a fenofibrate-induc-ible human apoAI transgene (32). Studies of PPAR  γ -specific agonists in mouse models of atherosclerosis have demonstrated protective effects in male mice that correlate with anti-inflam-matory effects in the artery wall and enhanced cholesterol efflux in cultured macrophages (31, 33–35). Furthermore, bone marrow transplantation of LDL receptor–deficient (LDLR  –/– ) mice with wild-type or PPAR  γ -knockout bone marrow pro-genitor cells demonstrated an antiatherogenic role of PPAR  γ  in macrophages (26). PPAR  β  agonists have not been evaluated in models of atherosclerosis, but bone marrow transplantation experiments in which LDLR  –/–  mice were reconstituted with PPAR  β –/–  bone marrow progenitor cells suggest that unliganded PPAR  β  can promote development of atherosclerosis (36).These previous findings support the concept that PPARs regu-late programs of gene expression that influence the development of atherosclerosis, but to our knowledge, the relative importance of proposed protective mechanisms have not been evaluated in  vivo. Here, we present a systematic analysis of the effects of spe-cific PPAR  α  and PPAR  β  agonists on the development of athero-sclerosis in male LDLR  –/–  mice, under conditions similar to those used previously in our laboratory to establish antiatherogenic effects of PPAR  γ  agonists. We examined the effects of specific agonists for all 3 PPAR subtypes on macrophage foam-cell forma-tion and inflammatory gene expression in vivo, and while all 3 PPAR agonists exerted potent anti-inflammatory effects in artery walls of hypercholesterolemic mice containing advanced lesions, only the PPAR  α  and PPAR  γ  agonists inhibited the development of atherosclerosis. Consistent with this, the PPAR  α  and PPAR  γ  agonists, but not the PPAR  β  agonist, inhibited the formation of macrophage foam cells in vivo. Unexpectedly, the present report of cholesterol uptake and efflux pathways in these cells suggests that PPAR  α  and PPAR  γ  agonists inhibit foam-cell formation at least in part through distinct  ABCA1-independent mechanisms. Results  Effects of PPAR  α  and PPAR  β  agonists on athero- sclerosis . To investigate potential effects of PPAR  α  and PPAR  β  on atherosclerosis, we performed intervention studies in LDLR  –/–  male mice using 2 potent receptor-spe-cific agonists under conditions in which we previously demonstrated that 2 PPAR  γ  agonists, rosiglitazone and GW7845, sig-nificantly inhibit lesion development (33). The PPAR  α -specific agonist GW7647 has a median effective concentration (EC 50 ) of 1 nM for the murine PPAR  α , compared to 2.9 μ M and 1.3 μ M for murine PPAR  β  and PPAR  γ , respectively (37). The PPAR  β  agonist GW0742 has an EC 50  of 28 nM for murine PPAR  β , versus 8.8 μ M and at least 10 μ M for murine PPAR  α  and PPAR  γ , respectively (38). LDLR  –/–  mice were fed a hypercholesterolemic diet with or without PPAR agonists for 14 weeks. Within 8 weeks, total plasma cholesterol reached approximately 2,000 mg/dl in the control, PPAR  α  agonist–, and PPAR  β  agonist–treated groups. Total cho-lesterol levels were not significantly different among the groups and a significant reduction in the triglyceride levels in mice treat-ed with the PPAR  β  agonist was only noted at the final time point (Table 1). HDL levels remained unchanged. No adverse health effects were noted throughout the study. Animals treated with the PPAR  α  agonist had a significantly higher (  P   < 0.001) liver to body weight ratio (0.079 ± 0.004, mean ± SEM) compared to ani-mals treated with the PPAR  β  agonist (0.057 ± 0.002) or to control animals (0.049 ± 0.004).The extent of atherosclerosis was determined in en face prepara-tions of the entire aorta after 14 weeks of the HC diet (Figure 1, A and B). Treatment with the PPAR  α  agonist yielded a 50% reduc-tion in atherosclerosis in the aortic arch and nearly a 90% reduc-tion in both the descending thoracic aorta (DTA) and abdominal aorta. In contrast, the extent of atherosclerosis in animals receiv-ing the PPAR  β  agonist was not significantly different from that of control animals in the arch or in the DTA. Atherosclerosis was also assessed in cross-sections through the aortic srcin (Figure 1, C and D). Consistent with the results throughout the aorta, animals that were fed the PPAR  α  agonist exhibited an approximately 50% reduction in cross-sectional lesion area compared to the control group. This effect was similar to the 40–70% reduction in lesion area previously observed for PPAR  γ  agonists under similar experi-mental conditions (33). Again, lesion size in animals fed the PPAR  β  agonist was not significantly different from that of controls.  Metabolic effects of PPAR  α  and PPAR  β  agonists . To investigate wheth-er metabolic differences might have contributed to the difference in antiatherogenic effect of the 2 agonists, we compared weight, insu-lin levels, and lipoprotein profiles (Figure 2). Animals fed the PPAR  α  agonist gained less weight compared with controls or animals fed the PPAR  β  agonist (Figure 2A), and they exhibited less adipose tis-sue. All animals consumed similar amounts of food throughout the study. Insulin levels were also significantly lower in animals treated with the PPAR  α  agonist (Figure 2B). Fast-performance liquid chro-matography (FPLC) analysis revealed a modest relative reduction Table 1 Total cholesterol, triglyceride, and HDL levels  0 Week 4 Weeks 8 Weeks 12 Weeks 14 WeeksTotal cholesterol (md/dl) Control ( n   = 8) 285 ± 28 1,486 ± 113 2,011 ± 198 1,950 ± 173 2,502 ± 193PPAR α  ligand ( n   = 9) 286 ± 11 1,424 ± 116 2,002 ± 135 2,013 ± 168 2,280 ± 168PPAR β  ligand ( n   = 10) 274 ± 18 1,387 ± 28 2,064 ± 64 2,113 ± 76 2,052 ± 175 Total triglycerides (mg/dl) Control 289 ± 28 248 ± 49 338 ± 94 349 ± 39 990 ± 156PPAR α  ligand 286 ± 11 221 ± 33 348 ± 78 313 ± 70 634 ± 62PPAR β  ligand 274 ± 18 167 ± 28 317 ± 64 420 ± 76 462 ± 175 A HDL (mg/dl )Control 189 ± 9PPAR α  ligand 201 ± 15PPAR β  ligand 195 ± 7 Data are expressed as mean ± SEM; n  represents the number of mice per group. Values were determined in plasma samples from fasting animals. A P  < 0.001.  research article 1566  The Journal of Clinical Investigation   http://www.jci.org Volume 114 Number 11 December 2004 in the VLDL, IDL/LDL, and HDL fractions in both of the treated groups, compared to the control group (Figure 2C).  Effects of PPAR  α  , β  , and γ  agonists on inflammatory gene expression . To examine the effects of PPAR agonists within the artery wall, we analyzed gene expression in animals with extensive atherosclerosis that were fed either the HC diet alone or the HC diet plus agonists for 14 weeks. All 3 PPAR agonists significantly inhibited the expres-sion of IFN- γ , TNF- α , MCP-1, VCAM-1, and ICAM-1, whereas less-er effects were observed for IL-1 β  (Figure 3). Experiments were also performed in younger mice with earlier lesions. These studies indi-cated some differences in the expression patterns of inflammatory markers and their responses to drug treatment. For example, IFN- γ  was not detected in earlier lesions, consistent with low absolute numbers of lymphocytes at this time point. MCP-1 levels, which were downregulated by all 3 PPAR agonists in late lesions, were actually upregulated in early lesions by the PPAR  α -specific agonist (data not shown), consistent with a previous report (16). Overall, however, these differences did not correlate with the effects of each PPAR agonist on the extent of atherosclerosis.  Influence of PPAR  α  , β  , and γ  agonists on genes regulating macrophage cho-lesterol metabolism in vivo . To investigate the effects of PPAR agonists on genes directly involved in foam-cell formation and cholesterol efflux in the artery wall, we used real-time PCR to measure mRNA levels of macrosialin, CD36, ABCA1, and LXR  α  in established atherosclerotic lesions (Figure 3). Aortas isolated from hypercholesterolemic mice exhibited a marked increase in macrosialin expression, consistent with an increased presence of intimal macrophages. As seen previ-ously, increased macrosialin expression was not accompanied by a concomitant increase in CD36 expression (33). Further increase of CD36 expression was observed only with the PPAR  γ  agonist. Expres-sion of ABCA1 also increased in hypercholesterolemic mice and was associated with the increased expression of LXR  α . However, none of the PPAR agonists altered the expression of ABCA1, even though the expression of LXR  α  was increased in mice treated with the PPAR  α  agonist. Although it is possible that ABCA1 is upregulated by PPAR ligands in a subset of cells within the aorta, these results suggest that upregulation of ABCA1 mRNA expression in the arterial wall is not the mechanism by which PPAR  α  and γ  agonists inhibit the develop-ment of atherosclerosis.  Influence of PPAR agonists on scavenger receptor activity in primary macrophages . To determine whether the effect of PPAR agonists on Figure 1 Atherosclerosis in LDLR –/–  male mice that were fed the HC diet for 14 weeks. ( A ) Sudan IV–stained en face preparations of aortas. Scale bars: 1 cm. ( B ) Quantitative analysis of atherosclerotic surface area in the entire aorta. ( C ) Sections through the aortic root at the level of the aortic valves. The micrographs are taken of sections at a similar distance from the aortic root. Original magnification, × 4. ( D ) Quanti-tative analysis of lesion areas in the aortic root. Data expressed as the mean ± SEM. C, control; α , PPAR α  agonist GW7647; β , PPAR β  agonist GW0742; Abd, abdominal aorta; Arch, aortic arch. * P  < 0.001 and ** P  ≤ 0.02, compared with control. Figure 2 Metabolic effects of PPAR ligands. Weight ( A ), plasma insulin levels ( B ), and size distribution ( C ) of lipoprotein particles fractionated by FPLC. Measurements of weight and plasma insulin levels were taken at the indicated time points. FPLC analysis of lipoproteins was done using pooled plasma from terminal bleeds. Circles, control; diamonds, PPAR α  agonist GW7647; squares, PPAR β  agonist GW0742. Data are expressed as mean ± SEM. * P  ≤ 0.05 compared with control. Mea-surements are from individual animals shown in Figure 1. Control, n  = 8; PPAR α  agonist, n  = 9; PPAR β  agonist, n  = 10.  research article  The Journal of Clinical Investigation   http://www.jci.org Volume 114 Number 11 December 2004 1567 foam-cell formation could be attributed to modulation of scav-enger-receptor activity, we measured uptake and degradation of oxLDL in isolated peritoneal macrophages treated in vitro with PPAR agonists for 24 hours (Figure 4A). As previously shown, the PPAR  γ  agonist significantly increased both uptake and deg-radation of oxLDL (20, 33), whereas treatment with the PPAR  α  agonist did not have a significant effect. The PPAR  β  agonist had a small but significant effect on the degradation of oxLDL. Effects of PPAR-specific agonists on oxLDL uptake and degradation were closely correlated with their effects on CD36 expression (Figure 4B). Scavenger receptor A (SRA) expression remained unchanged (Figure 4B). These findings suggest that the PPAR agonists do not inhibit foam-cell formation by downregulating scavenger recep-tors that mediate uptake of pathogenic lipoproteins.  Influence of PPAR agonists on cholesterol efflux pathways in cultured macrophages . Initial experiments were performed to evaluate effects of PPAR agonists on cholesterol efflux and expression of LXR  α  and ABCA1 in isolated peritoneal macrophages (Figure 4, C–F). The PPAR  γ  agonist, but not the PPAR  α  or β  agonists, stimulated apoAI-dependent cholesterol efflux to approximately the same extent as did the LXR agonist 24 (S), -25-epoxycholesterol (Fig-ure 4C). Consistent with these findings, the PPAR  γ  agonist, but not the PPAR  α  or β  agonists, stimulated expression of LXR  α  RNA (Figure 4D) and ABCA1 RNA and protein levels (Figure 4, E and F) in cholesterol-loaded macrophages.  Influence of PPAR agonists on macrophage foam-cell formation in vivo .  Although the ability of rosiglitazone to induce LXR  α  and ABCA1 expression in cultured macrophages is consistent with previous reports (27, 39), these responses were discrepant with the lack of an effect on the expression of LXR  α  and ABCA1 in the artery wall (Figure 3). We therefore developed an in vivo model for foam-cell formation by eliciting peritoneal macrophages in LDLR  –/–  mice that were fed an HC diet. In contrast to macrophages isolated from mice that were fed a normal chow diet, macrophages isolat-ed from hypercholesterolemic mice exhibited extensive Oil red O droplets (Figure 5A). Quantitative lipid analysis indicated a dra-matic increase in cholesteryl ester levels, accounting for the major change in neutral lipid content, as well as a modest increase in triglyceride content (Figure 5B). Treatment of animals with the PPAR  α  and PPAR  γ  agonists largely prevented lipid accumulation in these cells. In contrast, treatment with the PPAR  β  agonist did not reduce total cholesterol accumulation or overall oil red O staining, even though it decreased triglyceride levels and free cho-lesterol (Figure 5, A and B).These results were of particular interest because the effects of the 3 PPAR agonists on foam-cell formation within the peritoneal cavity paralleled their effects on the development of atheroscle-rotic lesions. All 3 PPAR subtypes were found to be expressed in peritoneal macrophages, although PPAR  α  expression was signifi-cantly lower than that of PPAR  β  or PPAR  γ  (Figure 5C). As in the case of primary macrophages treated with PPAR-specific agonists in vitro, CD36 expression was selectively increased in response to the PPAR  γ  agonist (Figure 5D). In contrast to results obtained in cultured macrophages, but consistent with findings in the artery wall, none of the PPAR agonists influenced the expression of  ABCA1 or LXR  α  in foam cells derived from the peritoneal cavity of hypercholesterolemic mice (Figure 5E). These observations sug-gest that PPAR agonists can inhibit foam-cell formation in vivo by LXR/ABCA1-independent mechanisms.  PPAR  α  agonists require macrophage expression of PPAR  α  to inhibit  foam-cell formation . To determine the roles of specific genes in PPAR-dependent inhibition of foam-cell formation, we developed and validated an assay based on the transfer of macrophages from donor animals into LDLR  –/–  mice. In preliminary experiments, elic-ited macrophages from wild-type donor animals were injected into the peritoneal cavities of LDLR  –/–  mice. Three days following injec-tion of 30 million donor cells, 10–15 million cells could be recov-ered from the peritoneal cavity of the recipient mouse. To deter-mine the fraction of donor cells recovered, donor macrophages bearing 20 copies of the λ gt11-  LacZ   transgene (40) were injected Figure 3 Gene expression in atherosclerotic lesions determined by real-time PCR analysis. Animals were fed an HC diet for 3 months (resulting in advanced lesions), followed by 4 weeks on the same diet supple-mented with the indicated PPAR agonists. Data are expressed as the mean ± SEM of triplicate measurements and are representative of 2 independent experiments. In each case, * P  ≤ 0.05, ** P  ≤ 0.01, and *** P  ≤ 0.001, compared with HC. Std, standard.  research article 1568  The Journal of Clinical Investigation   http://www.jci.org Volume 114 Number 11 December 2004 into recipient mice. This transgene is not normally expressed in mouse macrophages, and the gene itself therefore provides a spe-cific and quantitative marker of the transferred cells. Quantifica-tion of the lacZ   marker indicated that approximately 70% of the peritoneal macrophages that were recovered from recipient mice 3 days following transfer were of donor srcin (data not shown).Macrophages transferred from wild-type donors developed lipid droplets and increased cholesterol and triglyceride content when injected into LDLR  –/–  mice fed an HC diet (Figure 6, A and B), but not when injected into LDLR  –/–  recipients fed a normal diet (data not shown). The low levels of PPAR  α  mRNA in peritoneal macrophages (Figure 5C) raised the question of whether the inhibitory effects of PPAR  α -specific agonists on foam-cell formation in vivo were intrinsic to the macrophage. To address this question, we trans-ferred elicited macrophages from PPAR  α +/+  mice (both C57BL6/J and 129S backgrounds) and PPAR  α –/–  mice (129S background) into hypercholesterolemic LDLR  –/–  mice treated with or without the PPAR  α  agonist for 4 weeks. The PPAR  α  agonist inhibited foam-cell formation and dramatically reduced cholesteryl ester levels in macrophages derived from wild-type C57BL6 and 129S mice, but not in those from PPAR  α –/–  129S mice (Figure 6, A and B). These results indicate that the inhibitory effects of the PPAR  α  agonist are both PPAR  α  dependent and intrinsic to the macrophage. These data provide further support for the validity of the peritoneal cell trans-fer method, since the observed lack of effect of the PPAR  α -specific agonist in recipient animals receiving PPAR  α –/–  macrophages would not be expected if the majority of peritoneal cells were wild type (Fig-ure 6). Expression of ABCA1 in adoptively transferred PPAR  α +/+  and PPAR  –/–  macrophages was not altered by treatment with the PPAR  α  agonist, consistent with PPAR  α  regulating cholesterol homeostasis independently of ABCA1 (Figure 6C).  PPAR  α  agonists, but not PPAR  γ  agonists, require LXR to reduce foam-cell formation in peritoneal macrophages . To determine whether PPAR  α  and PPAR  γ  agonists can regulate foam-cell formation through an LXR-independent pathway, we performed macrophage transfer experiments using wild-type and LXR double knockout (DKO) mice. Following transfer into hypercholesterolemic LDLR  –/–  mice, LXR DKO macrophages stained more intensively with Oil red O and accumulated significantly more cholesteryl esters than did wild-type macrophages (Figure 7, A and B). These results are consistent with LXRs playing an important role in maintenance of cholesterol homeostasis. Triglyceride levels in the LXR DKO macrophages were also much lower, compared to untreated control macrophages, consistent with previous results demonstrating that LXRs play a role in triglyceride metabolism through SREBP-1 (41). As expected,  ABCA1 protein levels were reduced in macrophages derived from hypercholesterolemic LXR DKO animals, compared to levels in macrophages derived from hypercholesterolemic wild-type ani-mals (Figure 7C). However, treatment of recipient animals with the PPAR  γ  agonist led to significant reductions in oil red O staining and cholesterol content in both wild-type and LXR DKO macrophages (Figure 7, A and B). Similar results were observed using LXR  α –/–  macrophages (data not shown). Unexpectedly, when we transferred LXR DKO macrophages into LDLR  –/–  mice treated with the PPAR  α  agonist, we were unable to recover sufficient LXR DKO macrophages for subsequent analysis (data not shown), raising the possibility that activation of PPAR  α  exerted toxic effects in the absence of LXRs.To further investigate the role of LXRs in mediating inhibitory effects of PPAR  α  on foam-cell formation using an independent approach, we performed bone marrow transplantation experi-ments in which bone marrow from either wild-type or LXR DKO mice was used to reconstitute irradiated LDLR  –/–  mice. Recon-stituted mice were fed the HC diet and treated with PPAR  α  or PPAR  γ  agonists or control solvent prior to elicitation of peritoneal macrophages for analysis of foam-cell formation. Real-time PCR confirmed the absence of both LXR  α  and β  in these macrophages (Figure 7D). Treatment with the PPAR  γ  agonist rosiglitazone sig-nificantly inhibited foam-cell formation, consistent with results of the peritoneal macrophage transfer experiments. In contrast, treatment with the PPAR  α  agonist dramatically reduced the num-ber of viable LXR DKO macrophages that could be isolated from the peritoneal cavity, also consistent with the results of peritoneal macrophage transfer experiments. The few cells that were obtained Figure 4 Determination of scavenger receptor activity and cholesterol efflux in cultured peritoneal macrophages. PPAR-specific agonists, as indicated. ( A ) Influence of PPAR agonists on uptake (white bars) and degradation (black bars) of oxLDL. ( B ) Influence of PPAR agonists on CD36 and SRA expression by real-time PCR in hypercholesterolemic macrophages. ( C ) Influence of LXR and PPAR agonists on apoAI and HDL-specific cho-lesterol efflux in acLDL-loaded peritoneal macrophages. Expression of LXR α  ( D ) and ABCA1 ( E ) by real-time PCR in hypercholesterolemic macrophages treated with PPAR agonists in vitro. ( F ) Western blot analysis of ABCA1 protein in hypercholesterolemic macrophages treated with PPAR agonists as in E . Data are mean ± SEM and are representative of at least 2 independent experiments. In each case, * P  ≤ 0.05, ** P  ≤ 0.01, and *** P  < 0.001 compared with control. EPC, 24 (S), -25-epoxycholesterol; 125 I oxLDL, 125 I-labeled oxLDL.
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