Published online before print June 9, 2005, doi: 10.1161/01.ATV.0000173413.31789.1a
© 2005 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Conditional Knockout of Macrophage PPAR
Increases Atherosclerosis in C57BL/6 and Low-Density Lipoprotein Receptor–Deficient Mice
From the Departments of Medicine (V.R.B., P.G.Y., S.V.R., S.F., M.F.L.), Pharmacology (M.F.L.), Pathology (S.F.), Nephrology (V.K., M.D.B.), and Molecular Physiology & Biophysics (M.A.M.), Vanderbilt University Medical Center, Nashville, Tenn.
Correspondence to Vladimir Babaev, Sergio Fazio, or MacRae F. Linton, Department of Cardiovascular Medicine, Vanderbilt University School of Medicine, 312 PRB, Nashville, TN 37232-6300. E-mail vladimir.babaev{at}vanderbilt.edu
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Abstract |
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Objective— Peroxisome proliferator-activated receptor gamma (PPAR
) is highly expressed in macrophage-derived foam cells of atherosclerotic lesions, and its expression may have a dramatic impact on atherosclerosis.
Methods and Results— To investigate the contribution of macrophage PPAR expression on atherogenesis in vivo, we generated macrophage-specific PPAR knockout (MacPPARKO) mice. C57BL/6 and low-density lipoprotein (LDL) receptor–deficient (LDLR–/–) mice were reconstituted with MacPPARKO or wild-type marrow and challenged with an atherogenic diet. No differences were found in serum lipids between recipients reconstituted with MacPPARKO and wild-type marrow. In contrast, both C57BL/6 and LDLR–/– mice transplanted with MacPPARKO marrow had significantly larger atherosclerotic lesions than control recipients. In addition, MacPPARKO
LDLR–/– mice had higher numbers of macrophages in atherosclerotic lesions compared with controls. Peritoneal macrophages isolated from the MacPPARKO mice had decreased uptake of oxidized but not acetylated LDL and showed no changes in either cholesterol efflux or inflammatory cytokine expression. Macrophages from MacPPARKO mice had increased levels of migration and CC chemokine receptor 2 (CCR2) expression compared with wild-type macrophages.
Conclusion— Thus, macrophage PPAR deficiency increases atherosclerosis under conditions of mild and severe hypercholesterolemia, indicating an antiatherogenic role for PPAR, which may be caused, at least in part, by modulation of CCR2 expression and monocyte recruitment.
To investigate the contribution of macrophage PPAR on atherogenesis, we generated macrophage-specific PPAR knockout (MacPPARKO) mice. C57BL/6 and low-density lipoprotein (LDL) receptor–deficient mice were reconstituted with MacPPARKO marrow. These mice had significantly larger atherosclerotic lesions than control recipients. MacPPARKO macrophages had decreased uptake of oxidized LDL and increased CCR2 expression levels.
Key Words: ABCA1 • atherosclerosis • CCR2 expression • cholesterol efflux • macrophages • scavenger receptor CD36
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Introduction |
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Peroxisome proliferator-activated receptor gamma (PPAR
) is a nuclear transcription factor that regulates a large number of genes important in lipid metabolism and inflammation.1 The receptor is highly expressed in macrophages and macrophage-derived foam cells of atherosclerotic lesions,2–4 and its expression may critically affect macrophage functions that impact atherosclerosis, including activation, cytokine production, recruitment, and transformation into foam cells.
Several studies have shown that the administration of PPAR agonists inhibits the development of atherosclerosis in low-density lipoprotein (LDL) receptor–deficient (LDLR–/–)5,6 and apolipoprotein E-deficient (apoE–/–) mice.7 Consistent with this, mice transplanted with bone marrow from a PPAR–/– chimera mouse exhibit a significant increase in atherosclerosis.8 These data all support an antiatherogenic role for macrophage PPAR in atherosclerotic lesion development.
It has been assumed that the antiatherogenic effects of macrophage PPAR expression may derive from activation of genes responsible for cholesterol efflux, thus shifting the balance from lipid loading to lipid efflux.8 Recent studies, however, have not confirmed the role of PPAR ligands in cholesterol efflux by macrophages.9,10 PPAR may also exert antiinflammatory effects in macrophages directly,11 or through LXR
12 by negatively interfering with the AP-1, NF
B, and STAT signaling pathways,13 or by reducing tumor necrosis factor-, IL-1, and IL-6 secretion.14 However, the loss of PPAR expression in macrophages derived from embryonic stem cells does not appear to alter basal or stimulated levels of cytokine secretion.15,16
In vitro studies have demonstrated that PPAR expression by human and murine monocytes directly inhibits CC chemokine receptor 2 (CCR2) expression and suppresses MCP-1–mediated chemotaxis.17 In addition, pretreatment of monocytes with PPAR agonists reduced their adhesion to vascular endothelium18 and their transendothelial migration.6 Based on these data, it appears that at least some of the antiatherogenic effects of macrophage PPAR may be attributed to inhibition of macrophage recruitment and migration; however, the physiological relevance of the inhibitory processes in vivo remains unclear.
Targeted disruption of the PPAR gene in mice causes early embryonic lethality19–21 and thus presents an obstacle to a systematic study of the gene’s role in atherogenesis. Therefore, we generated mice with a macrophage-specific PPAR knockout (MacPPARKO) using the Cre-loxP recombination system approach under the control of the murine M lysozyme promoter. C57BL6 and LDLR–/– mice were lethally irradiated, reconstituted with marrow from MacPPARKO or wild-type mice, and challenged with atherogenic diets. Mice reconstituted with MacPPARKO marrow exhibited significantly larger atherosclerotic lesions with increased numbers of macrophages. Macrophages from these mice also expressed higher levels of CCR2, suggesting that an increase in monocyte recruitment may be responsible for the accelerated atherosclerosis seen in these mice.
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Methods |
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Animal Procedures
Mice with the "floxed" PPAR
(PPARfl/fl) gene,22 a mouse Cre line under the control of the murine M lysozyme promoter,23 and transgenic ROSA26R24 were at the sixth or more backcross into C57BL/6 background. ROSA26 mice,25 recipient C57BL/6, and LDLR–/– mice on C57BL/6 background were purchased from Jackson Laboratories Inc (Bar Harbor, Me). All mice were maintained in micro-isolator cages on a rodent chow diet containing 4.5% fat (PMI number 5010) and autoclaved acidified (pH 2.8) water. A butterfat diet contained 19.5% butterfat, 1.25% cholesterol, and 0.5% cholic acid (ICN, Avrora, Ohio). A Western diet consisted of 21% fat and 0.15% cholesterol (Teklad). Animal care and experimental procedures were performed according to the regulations of Vanderbilt University’s Animal Care Committee.
Bone Marrow Transplantation
Recipient mice were lethally irradiated (9 Gy) from a cesium gamma source, and 5x106 bone marrow cells were injected as described.26
Flow Cytometry
To examine lacZ gene expression, peritoneal macrophages were treated with ß-galactosidase-fluorescein di-ß-D-galactopyranoside (Molecular Probes, Eugene, Ore) as described.26 To detect CCR2 expression, macrophages were incubated with nonspecific mouse IgG (Sigma), then with allophycocyanin (activated protein C)-conjugated mouse anti-human CCR2 (R&D Systems Inc, Minneapolis, Minn) and analyzed by flow cytometry (Becton Dickinson).
Serum Lipids and Lipoprotein Profiles
Mice were fasted for 4 hours, and the serum total cholesterol and triglycerides were determined as described.27 Fast performance liquid chromatography (fast protein liquid) was completed using a Superose 6 column (Pharmacia) on a high-performance liquid chromatography system model 600 (Waters).
Analysis of Aortic Lesion
The aorta was flushed through the left ventricle and dissected for en face preparation and image analysis. Cryosections of the proximal aorta were prepared and analyzed using an Imaging system KS 300 (Kontron Electronik GmbH) as described.28
Modified LDL Uptake
Thioglycolate-elicited peritoneal macrophages were cultured in DMEM with 10% fetal bovine serum for 2 days. Then cells were incubated with DiI-labeled human acetylated LDL (AcLDL) or oxidized LDL (Intracel Corp, Rockville, Md) at 37°C for 4 hours and analyzed under a fluorescent microscope or by fluorescence-activated cell sorter (FACS) flow cytometry.16
Cholesterol Loading and Efflux
Macrophages were cultured in DMEM containing 1% fetal bovine serum, 3 µCi/mL of 3H-cholesterol, and 70 µg/mL of human AcLDL for 48 hours. Cholesterol pools were equilibrated overnight in 0.1% bovine serum albumin–DMEM. Then cells were incubated with human apolipoprotein AI (20 µg/mL) or high-density lipoprotein (50 µg/mL) for up to 7 hours. For each time point, 3H-cholesterol was measured in aliquots of media. The cell lipids were extracted and used for measurement of 3H cholesterol.
Ligand Treatment and Real-Time Polymerase Chain Reaction
Macrophages were cultured in DMEM media supplemented with 5% lipoprotein-deficient fetal bovine serum (Intracel) with or without 10 µmol/L per mL rosiglitazone for 24 hours. Total RNA was isolated from peritoneal macrophages using the Trizol reagent (Invitrogen, Carlsbad, Calif) purified by RNA Easy kit (Qiagen). Relative quantitation of the target mRNA were performed on the ABI Prism 7000 Sequence Detection System (Applied Biosystems) and normalized to ß-actin or 18S ribosomal RNA. Probes for PPAR, CD36, apoE, ABCA1, MCP-1, Gro1, and CCR2 were provided by Applied Biosystems.
Migration Assay
In vitro assays were performed in a 96-well modified Boyden chamber with a 3-µm filter pore size (Millipore). Cell solution (100 µL) was added to each well in the top filter plate portion of the assembly, and MCP-1 (0.1 µg/µL) or media was added to the bottom feeder wells. After 1 hour, the upper portion was removed and cell numbers were counted.
Statistical Analysis
The statistical differences in mean serum lipids and aortic lesion areas between the groups were determined using the SigmaStat V.2 software (SPSS Inc, Chicago, Ill) by Student t test and the Mann–Whitney rank sum test, respectively.
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Results |
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Macrophage-Specific PPAR
KnockoutUsing the Cre-loxP recombination system, we generated MacPPAR
KO mice on the C57BL/6 background. These mice were viable and fertile with no notable differences in body weight or plasma lipid levels when compared with PPARfl/fl littermates. The level of PPAR RNA was dramatically decreased in peritoneal macrophages from MacPPARKOmice compared with that of wild-type macrophages as analyzed by RT-PCR (Figure 1A) or quantitative real-time PCR normalized to 18S ribosomal RNA (Figure 1B). Deficiency in PPAR RNA was specific for macrophages and not observed in kidney, adipose, or liver tissue (data not shown).
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) and MacPPAR
) macrophage analyzed by real-time quantitative PCR. LacZ expression is detected by flow cytometry in peritoneal macrophages isolated from PPAR
To monitor the effectiveness of Cre recombinase, we crossed MacPPARKO mice with transgenic ROSA26R mice. This strain has a "floxed" STOP codon at the 5' end of the lacZ gene driven by the ß-actin promoter.24 In MacPPARKO/ROSA26R mice, Cre recombinase excised the STOP codon, releasing lacZ expression in the majority (93% to 96%) of peritoneal macrophages. The intensity of lacZ expression in these cells, as measured by a FACS assay, was significantly higher when compared with macrophages isolated from PPARfl/fl and ROSA26 mice ubiquitously expressing the lacZ gene (Figure 1C).
Role of Macrophage PPAR in Atherosclerosis
To examine the impact of MacPPARKO on atherosclerosis, 7-week-old female C57BL/6 and 8-week-old female LDLR–/– mice were lethally irradiated and transplanted with marrow from female MacPPARKO (n=16 for each experimental group) or PPARfl/fl mice (n=15 for each control group). Six or 4 weeks after transplantation, recipient mice were challenged with the butterfat or the Western diets for 16 or 8 weeks, respectively.
Serum lipid levels did not differ significantly between the control and experimental groups of mice on either the chow or the atherogenic diets with exception that the triglycerides were higher in MacPPARKOC57BL/6 mice after 12 weeks of the butterfat diet (Tables I and II, available online at http://atvb.ahajournals.org). Similarly, serum lipoprotein profiles did not differ significantly between experimental and control groups of the recipient mice (Figure Ia and Ib, available online at http://atvb.ahajournals.org). In contrast, the extent of atherosclerotic lesions in the proximal aortas of C57BL/6 and LDLR–/– recipients reconstituted with MacPPARKO macrophages was significantly greater (48% and 84%) compared with PPARfl/flC57BL/6 (37 715±3010 versus 25 512±2660 µm2; P=0.005) and PPARfl/flLDLR–/– mice (125 120±9550 versus 67 948±6205 µm2; P<0.001), respectively (Figure 2A and 2B). MacPPARKOLDLR–/– recipients had larger (46%) lesion area in the distal aortas analyzed en face compared with PPARfl/flLDLR–/– mice (0.19± 0.01 versus 0.13±0.02%; P=0.031; Figure 2C). Thus, macrophage PPAR expression plays a protective role in atherosclerotic lesion formation.
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) marrow after 16 or 8 weeks on their atherogenic diets, respectively. The extent of atherosclerosis in "pinned out" aortas en face of LDLR–/– (C) mice.
Modified LDL Uptake and Cholesterol Efflux by Macrophages From MacPPARKO Mice
In an effort to better understand the molecular basis of these effects, we first analyzed the impact of the macrophage PPAR gene deletion on uptake of modified LDL. Peritoneal macrophages were isolated and incubated with DiI-labeled oxidized and acetylated LDL. Microscopic analysis showed that PPAR–/– macrophages accumulated significantly less oxidized LDL than wild-type macrophages (Figure 3A). FACS analysis demonstrated that PPAR–/– macrophages had a reduced (43% to 57%) levels of oxidized LDL uptake but not AcLDL uptake, compared with wild-type macrophages (Figure 3B and 3C). To test cholesterol efflux, macrophages were loaded with human AcLDL cholesterol and incubated with high-density lipoprotein or apolipoprotein AI. No differences were noted in either high-density lipoprotein- or apolipoprotein AI-mediated cholesterol efflux from PPAR–/– and wild-type macrophages (Figure 4A and 4B). However, real-time PCR analysis revealed that wild-type macrophages treated with a PPAR ligand, rosiglitazone, expressed significantly higher levels of CD36 (269%) and ABCA1 (125%) but not apoE RNA compared with control nontreated cells (Figure 4C to 4F). In PPAR–/– macrophages, these stimulation effects for the CD36 and ABCA1 genes were lost.
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Inflammatory Cytokine Gene Profiles and CCR2 Expression by Macrophages From MacPPARKO Mice
To examine the antiinflammatory effects of macrophage PPAR, mRNA was isolated from lipopolysaccharide-activated macrophages and analyzed using an inflammatory response cytokines gene array kit. For both type of macrophages, expression levels for the majority of cytokines (IL1a, IL1b, IL6, IL12a, IL18, transforming growth factor-ß, and tumor necrosis factor-) were not significantly different, with the exception of the Gro1 oncogene (Table III, available online at http://atvb.ahajournals.org). As confirmed by real-time PCR, PPAR–/– macrophages stimulated by lipopolysaccharide had increased levels of the Gro1 (1.7 fold) but not MCP-1 expression compared with wild-type macrophages (Figure IVa and IVb, available online at http://atvb.ahajournals.org).
Finally, the impact of PPAR expression by macrophages on the CCR2/MCP-1 pathway was analyzed by real-time PCR. The level of mRNA CCR2 expression was significantly increased (1.7-fold) in PPAR–/– macrophages compared with wild-type macrophages (Figure 5A). In addition, PPAR–/– macrophages had increased levels CCR2 protein expression (107±12 versus 75±5; P<0.05) as detected by FACS (Figure 5B).
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Given the pivotal role of the CCR2 pathway in monocyte recruitment, we performed a series of in vitro experiments to determine the ability of peritoneal macrophages to migrate. Macrophages from MacPPARKO mice migrated significantly faster in both nonstimulated and MCP-1–directed tests compared with macrophages from PPARfl/fl mice (Figure 5C). In addition, we stained sections from the proximal aorta of LDLR–/– recipients using a macrophage-specific antibody and DAPI (Figure 6). MacPPARKOLDLR–/– mice had a significantly increased (36%) number of macrophages per a section (195±14) compared with control PPARfl/flLDLR–/– mice (143±6; P<0.004).
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Discussion |
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To examine the role of macrophage PPAR
expression in atherosclerotic lesion formation, we generated mice with MacPPARKO using the Cre-LoxP recombination system under control of the murine M lysozyme promoter. Then, C57BL6 and LDLR–/– mice were reconstituted with MacPPARKO or wild-type macrophages and fed atherogenic diets. The serum lipid levels and lipoprotein profiles were similar between control and experimental groups of recipients. In contrast, mice reconstituted with MacPPARKO macrophages exhibited significantly larger atherosclerotic lesions compared with controls. Thus, local expression of macrophage PPAR in artery walls plays a protective antiatherogenic role under conditions of mild and severe hypercholesterolemia.
In these studies, we have used a novel macrophage-specific knockout approach to examine the role of macrophage PPAR in atherogenesis. To our knowledge, this is the first report of the Lys-M-Cre approach to examine the effects of macrophage-specific gene expression in atherosclerosis in vivo. Although Akiyama et al10 previously developed a macrophage-specific knockout of PPAR driven by the MX1 promoter, they did not report the impact of PPAR deficiency on atherosclerosis. Furthermore, our approach has an advantage over the MX1 promoter approach in that the LysM-Cre mice do not need induction to develop PPAR deficiency. Chawla et al8 reported similar antiatherogenic effects of macrophage PPAR expression in LDLR–/– mice reconstituted with PPAR–/– marrow from a chimeric mouse. Consistent with the results of genetic deletion of macrophage PPAR, administration of PPAR agonists to LDLR–/– mice5,6 and apoE–/– mice,7 and in balloon injury experiments,29 have also demonstrated an antiatherogenic role for PPAR.
To investigate possible mechanisms by which macrophage PPAR delivers its antiatherogenic effects, we first focused on the ability of macrophages to take-up modified lipoproteins. We found that MacPPARKO macrophages have decreased uptake of oxidized but not acetylated LDL and, unlike wild-type macrophages, did not show an increase in CD36 expression in response treatment with PPAR agonists. These findings are consistent with previous ex vivo studies demonstrating that PPAR has a critical role in the basal regulation of the CD36 gene in macrophages.4 Interestingly, Liang et al have recently reported that, in the setting of extreme insulin resistance caused by leptin deficiency found in ob/ob and ob/ob LDLR–/– mice, treatment with a thiazolidinedione results in reduced systemic insulin resistance leading to reduced macrophage CD36 protein, despite an increase in macrophage CD36 gene expression, caused by correction of defective insulin signaling in the macrophage.30 In contrast, in vivo thiazoladinedione treatment of LDLR–/– mice on a Western diet, a model associated with mild insulin resistance, resulted in a 3-fold increase in macrophage CD36 expression.30 Thus, the impact of PPAR agonists on macrophage CD36 protein may vary with the degree of insulin resistance. Given that PPAR agonists have been reported to reduce atherosclerosis in LDLR-deficient mice,5,6 the potentially proatherogenic effects of CD36 upregulation on foam cell formation15,31 are apparently outweighed by other antiatherogenic effects of PPAR agonists in this model.
Next, we found that PPAR–/– and wild-type macrophages have similar basal levels of cholesterol efflux. However, the agonist treatment increased ABCAI gene expression levels in wild-type but not in PPAR–/– macrophages, consistent with previous studies suggesting that the PPAR-LXR-ABCA1 pathway may be important in modulating the development of atherosclerosis.8,32
Macrophage PPAR mediates the activation of a large number of genes that are important in inflammation.33 To test how PPAR deficiency affects the macrophage’s ability to produce cytokines in response to lipopolysaccharide, inflammatory cytokine gene expression profiles were compared in macrophages from MacPPARKO and wild-type mice. The majority of cytokines had similar levels of expression for both groups of macrophages, indicating that at least some of the previously described effects of PPAR agonists on cytokine gene expression are independent of PPAR gene expression.15,16 At the same time, MacPPARKOLDLR–/– mice had increased macrophage numbers in atherosclerotic lesions, augmented macrophage CCR2 expression, and migration. These data suggest that PPAR modulates CCR2 expression and may affect monocyte recruitment.
Monocyte CCR2 expression is increased in hypercholesterolemic patients.34 Native LDL and oxidative stress increase CCR2 gene expression, whereas antioxidants rapidly inhibit it.35 Treatment with oxidized LDL activates PPAR expression and PPAR agonists markedly attenuate CCR2 expression in circulating monocytes.17 Recent in vivo studies have shown that PPAR activators suppress the recruitment of inflammatory cells via a PPAR-dependent mechanism in cases of experimental glomerulonephritis36 and myocardial infarction in rats.37 All these data support the concept that PPAR-modulated CCR2 expression may impact the development of atherosclerosis through an effect on monocyte recruitment.
Mounting evidence suggests that the MCP-1/CCR2 pathway is important in atherogenesis. Targeted deletion of CCR2 or its ligand MCP-1 significantly decreased macrophage recruitment and atherosclerotic lesion size in apoE–/– mice.38–40 Clinical studies also demonstrated that coronary atherosclerosis is decreased in patients with a polymorphism of the CCR2 gene that reduces its function.41 In addition, PPAR agonists inhibit CCR2 expression in monocytes and atherosclerosis development in rats.42,43 Thus, PPAR-mediated CCR2 expression by macrophages may be an important pathway in atherogenesis and provides a novel therapeutic target for prevention or treatment of atherosclerosis.
The role of macrophage PPAR in atherosclerosis is clearly complex and likely includes important effects on cholesterol homeostasis and inflammatory pathways, which may vary with lesion stage and metabolic factors such as insulin resistance.30 Our conditional macrophage-specific knockout of PPAR presents a new opportunity to study the role of macrophage PPAR gene expression in atherogenesis in vivo. C57BL6 and LDLR–/– mice reconstituted with PPAR–/– macrophages developed significantly larger atherosclerotic lesions compared with control mice in response to atherogenic diets. In the absence of any notable changes in serum lipids between control and experimental mice, the increase in atherosclerosis suggests that macrophage PPAR is crucial for these antiatherogenic effects. The increase in CCR2 expression by macrophages from MacPPARKO mice suggests a novel role for PPAR in monocyte recruitment and the development of atherosclerosis.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL65405, HL53989, HL 57986, DK59637 (Lipid, Lipoprotein, and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Centers). V.R.B. is supported by an American Heart Association grant (0160160B). The authors thank Lei Ding and Youmin Zhang for excellent technical expertise.
Received January 5, 2005; accepted May 27, 2005.
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