Published online before print August 27, 2007, doi: 10.1161/CIRCULATIONAHA.106.684704
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2007;116:1404-1412.)
© 2007 American Heart Association, Inc.
Vascular Medicine |
Macrophage Expression of Peroxisome Proliferator–Activated Receptor-
Reduces Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice
From the Department of Medicine (V.R.B., H.I., L.D., P.G.Y., D.E.D., S.F., M.F.L.), Pathology (S.F.), and Pharmacology (M.F.L.), Vanderbilt University Medical Center, Nashville, Tenn; Division of Endocrinology and Metabolism (W.J.K.), University of Texas, Dallas, Tex; and Washington University (C.F.S.), St. Louis, Mo.
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, sergio.fazio@vanderbilt.edu, or macrae.linton@vanderbilt.edu
Received December 15, 2006; accepted July 12, 2007.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— The peroxisome proliferator–activated receptor-
(PPAR) plays important roles in lipid metabolism, inflammation, and atherosclerosis. PPAR ligands have been shown to reduce cardiovascular events in high-risk subjects. PPAR expression by arterial cells, including macrophages, may exert local antiatherogenic effects independent of plasma lipid changes.
Methods and Results— To examine the contribution of PPAR expression by bone marrow–derived cells in atherosclerosis, male and female low-density lipoprotein receptor–deficient (LDLR–/–) mice were reconstituted with bone marrow from PPAR–/– or PPAR+/+ mice and challenged with a high-fat diet. Although serum lipids and lipoprotein profiles did not differ between the groups, the size of atherosclerotic lesions in the distal aorta of male and female PPAR–/–
LDLR–/– mice was significantly increased (44% and 46%, respectively) compared with controls. Male PPAR–/–LDLR–/– mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPAR+/+LDLR–/– mice. Peritoneal macrophages from PPAR–/– mice had increased uptake of oxidized LDL and decreased cholesterol efflux. PPAR–/– macrophages had lower levels of scavenger receptor B type I and ABCA1 protein expression and an accelerated response of nuclear factor-
B–regulated inflammatory genes. A laser capture microdissection analysis verified suppressed scavenger receptor B type I and increased nuclear factor-B gene expression levels in vivo in atherosclerotic lesions of PPAR–/–LDLR–/– mice compared with the lesions of control PPAR+/+LDLR–/– mice.
Conclusions— These data demonstrate that PPAR expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.
Key Words: atherosclerosis • macrophages • proteins • receptors
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Peroxisome proliferator–activated receptor-alpha (PPAR
) is a ligand-activated transcription factor that plays an important role in lipid metabolism and inflammation. PPAR regulates a number of genes involved in inflammation and the metabolism of cellular lipids, plasma lipoproteins, and glucose.1,2
Clinical Perspective p 1412
PPAR ligands, such as fibric acid derivates, have widespread clinical application in the treatment of dyslipidemia.3 They have been shown to reduce the progression of coronary disease in angiographic trials4,5 and to reduce the incidence of cardiovascular events in outcome studies.6–8 The protective effects of PPAR agonists on atherosclerosis may be due to their impact on both inflammation and cholesterol homeostasis in the artery wall.9 It was recently shown that a PPAR gene polymorphism significantly affects progression of atherosclerosis and ischemic heart disease without causing changes in plasma cholesterol levels.10
Studies of the role of PPAR in atherosclerosis in murine models using PPAR agonists and knockout mice have yielded conflicting results. Tordjman et al11 showed that mice with double deletion of the PPAR and apolipoprotein E (apoE) genes had higher levels of atherogenic lipoproteins but less aortic atherosclerosis, improved insulin sensitivity, and significantly lower levels of blood pressure than PPAR+/+/apoE–/– litter mates, which suggests that systemic PPAR deficiency has complex effects on atherogenesis. In one study, treatment of apoE–/– mice with PPAR agonists severely aggravated their hypercholesterolemia and accelerated atherosclerotic development.12 In contrast, several reports have demonstrated that treatment with PPAR agonists improved lipoprotein metabolism and reduced atherosclerosis in low-density lipoprotein receptor (LDL)–deficient (LDLR–/–), apolipoprotein E–deficient (apoE–/–), and human apoE2–expressing (instead of mouse apoE–expressing) mice.13–15 In addition, PPAR agonists have been reported to reduce cholesterol esterification in macrophages.16 Taken together, these data demonstrate that the effects of PPAR on atherogenesis are complex and may vary in different conditions.
In atherosclerotic lesions, PPAR is expressed by endothelial cells,17 smooth muscle cells,18 and monocyte/macrophages.19 To dissect the role of macrophage PPAR expression in early atherosclerosis, we generated female and male LDLR–/– mice chimeric for PPAR–/– macrophages. When fed the Western diet, recipient mice reconstituted with PPAR–/– bone marrow–derived cells developed significantly larger atherosclerotic lesions than control mice with wild-type marrow. Peritoneal macrophages isolated from PPAR–/– mice had slightly decreased cholesterol efflux, increased uptake of oxidized LDL, and accelerated response of proinflammatory genes. These data together demonstrate that PPAR expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animal Procedures
Mice with targeted disruption of the PPAR
gene20 were crossed with LDLR–/– mice21 onto the C57BL/6 background (fifth backcross). Recipient LDLR–/– mice on the C57BL/6 background were purchased from Jackson Laboratories Inc (Bar Harbor, Me). All mice were maintained on a rodent chow diet (PMI No. 5010, St. Louis, Mo) or the Western-type diet consisting of 21% fat and 0.15% cholesterol (Teklad, Madison, Wis). 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 previously.22
Serum Lipids and Lipoprotein Distribution
Mice were fasted for 4 hours, and then serum total cholesterol and triglyceride levels were determined.23 Fast-performance liquid chromatography was performed with a Superose 6 column (Pharmacia, Piscataway, NJ) on a high-performance liquid chromatography (HPLC) system model 600 (Waters, Milford, Mass). High-density lipoprotein (HDL) cholesterol was measured on an automated ACE analyzer with the direct HDL test (Schiapparelli Biosystems, Inc, Fairfield, NJ).
Analysis of Aortic Lesions
The entire aorta was flushed and dissected for en face preparation.24 Sections of the proximal aorta were prepared25 and analyzed with an imaging system KS 300 (Carl Zeiss Inc, Thornwood, NY).
Modified LDL Uptake and Cholesterol Efflux Measurements
Thioglycolate-elicited peritoneal macrophages were cultured for 2 days, incubated with human 1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)–labeled acetylated or oxidized LDL (Intracel Corp, Rockville, Md) for 4 hours, and examined by flow cytometry as described previously.26 For cholesterol loading, macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 2% fetal calf serum containing 1.5 to 2.0 µCi/mL [3H]-cholesterol and 70 µg/mL human acetylated LDL for 36 hours. The cells were incubated with human HDL (100 µg/mL) for 24 hours, then radioactivity was measured in media, and lipids were extracted with hexamine-isopropanol. Cholesterol efflux was expressed as the percentage of the counts in the cellular lysate. In a separate set of experiments, the cells were incubated in DMEM containing 0.25% FBS that contained human acetylated LDL (AcLDL; 70 µg/mL) for 48 hours. The cells were equilibrated in DMEM containing 1% BSA for 18 hours and then treated with DMEM alone or with human HDL (100 µg/mL; Intracel, Frederick, Md) for 24 and 48 hours. Cell lipids were extracted by overnight incubation at room temperature in isopropanol containing cholesteryl methyl ether (2.5 µg/well) as an internal standard. Total cholesterol of lipid extracts was measured by gas-liquid chromatography after the procedure.27
Quantitative Real-Time Polymerase Chain Reaction
Peritoneal macrophages were treated with RPMI media containing 5% fetal lipoprotein-deficient serum (Intracel) and 2 PPAR agonists, WY14643 (Biomol Res Lab Inc, Plymouth Meeting, Pa) (2 µmol/L) or GW7647 (Calbiochem, San Diego, Calif) (600 nmol/L), for 24 hours. In addition, some cells were treated with lipopolysaccharide (LPS; 50 ng/mL; Sigma, St. Louis, Mo) for 5 hours. Total RNA was isolated from peritoneal macrophages with Trizol reagent (Invitrogen, Carlsbad, Calif) purified by RNeasy kit (Qiagen, Valencia, Calif). Relative quantitation of the target mRNA was performed on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, Calif) and normalized with 18S RNA as an endogenous control as described previously.28 All probes were purchased from Applied Biosystems.
Western Blotting
Proteins were extracted from macrophages in the presence of protease inhibitor (Sigma) and resolved (100 µg/well) in NuPAGE 4% to 12% Bis-Tris gel (Invitrogen) and then blotted onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). Proteins were detected with rabbit antibodies to scavenger receptor B type I (SR-BI), ABCA1 (both from Novus Biologicals, Littleton, Colo), and ß-actin (Abcam, Inc, Cambridge, Mass). Immunoreactive bands were visualized with horseradish peroxidase–conjugated goat anti-rabbit antibodies (Upstate Cell Signaling, Lake Placid, NY) and quantified by densitometry.
Laser Capture Microdissection
Cryosections (7 µm) from the proximal aorta were used for laser capture microdissection (LCM) as reported previously.29 Serial sections were stained by specific anti-macrophage antibody, MOMA-2 (Serotec, Raleigh, NC). Macrophage-rich atherosclerotic lesions were captured (PixCell II LCM system; Arcturus, Mountain View, Calif) from the proximal aorta of mice reconstituted with wild-type and PPARa–/– marrow (n=5 in each group). Approximately 500 shots were performed with a 15-µm diameter infrared laser beam to obtain approximately 200 to 1500 cells of interest (CapSure LCM caps; Arcturus Biosciences). Total mRNA concentration was measured by a NanoDrop ND-1000 spectrophotometer (Wilmington, Del), and its purity was analyzed by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif) with the DNA markers. RNA was isolated from captured tissues and amplified (PicoPure RNA isolation and amplification kits, Arcturus).
Statistical Analysis
The statistical differences in mean serum lipids and aortic lesion areas between the groups were determined with SigmaStat version 2 software (SPSS Inc, Chicago, Ill) by Student t test and the Mann-Whitney rank sum test, respectively.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Macrophage PPAR
Deficiency and Serum LipidsTo study the role of PPAR
expression by bone marrow–derived cells in atherosclerosis, 2 main sets of experiments were conducted. First, 7-week-old female LDLR–/– mice were lethally irradiated and reconstituted with marrow from female PPAR–/–/LDLR–/– (n=12, experimental group) or PPARa+/+/LDLR–/– (n=12, control group) mice. Second, 7-week-old male LDLR–/– mice were lethally irradiated and reconstituted with marrow from male PPAR–/–/LDLR–/– (n=15) or PPARa+/+/LDLR–/– (n=13) mice. After 6 weeks on chow diet, the recipient mice were challenged with the Western diet for 10 and 8 weeks, respectively. Female but not male PPAR–/–LDLR–/– mice gained more body weight than control PPAR+/+LDLR–/– mice (30.8±1.0 versus 27.1±0.6 g, mean±SEM; P<0.043). Female and male recipients had no changes in systolic blood pressure or plasma postheparin lipoprotein lipase activity (data not shown). Serum cholesterol and triglycerides did not differ between the control and experimental groups in the course of the diet (Table). Lipoprotein profiles (Figure 1A and 1B) and HDL cholesterol levels in female and male PPAR–/–LDLR–/– mice were similar to those of control recipients (208±12 versus 196±11 mg/dL, P=0.34, and 164±16 versus 164±13 mg/dL, P=0.99, respectively). Thus, PPAR deficiency in bone marrow–derived cells has no impact on serum lipids and lipoprotein profiles.
|
|
) bone marrow after 10 and 8 weeks on the Western diet, respectively. Data are represented as the average (n=3) of the percent of total cholesterol for each fraction. Fractions 14 to 17 contain very low-density lipoprotein (VLDL); fractions 18 to 24 are intermediate-density lipoprotein/LDL; and fractions 25 to 30 contain HDL.
Macrophage PPAR Deficiency and Atherosclerosis
After 10 and 8 weeks on the diet, aortic lesions were detected in all female and male recipients. These lesions consisted mainly of macrophage-derived foam cells, as verified by analysis of the sections stained for macrophages (data not shown). Atherosclerotic lesions in female recipients often contained a fibrous cap and had a lower ratio of macrophage area to oil red O staining area (0.50 versus 0.53, respectively) than male recipients (0.68 versus 0.69, respectively). In male PPAR–/–LDLR–/– mice, the extent of atherosclerotic lesions in the proximal aorta was significantly higher (44%) than in PPAR+/+LDLR–/– mice (90 127±9690 versus 62 648±7424 µm2, P=0.048; Figure 2D). A similar increase in atherosclerotic lesions was found in en face aorta preparations of male PPAR–/–LDLR–/– mice compared with PPAR+/+LDLR–/– male recipients (0.13±0.015% versus 0.09±0.01%, P=0.044; Figure 2C). In contrast, no difference in lesion size existed in the proximal aorta of female PPAR–/–LDLR–/– and control PPAR+/+LDLR–/– recipients (15 3763±19 932 versus 14 8036±20 770 µm2, P=0.85; Figure 2A). However, a significant 46% increase in lesion area was noted by en face analysis of the distal aorta of female PPAR–/–LDLR–/–mice compared with PPAR+/+LDLR–/– mice (0.41±0.06% versus 0.28±0.02%, respectively, P<0.043; Figure 2B). Thus, PPAR deficiency in marrow-derived cells significantly increased atherosclerosis in both female and male LDLR–/– mice.
|
Oxidized LDL Uptake and Cholesterol Efflux by PPAR–/– Macrophages
To study the impact of PPAR on the uptake of modified LDL, macrophages from PPAR–/– and PPAR+/+ mice were incubated with DiI-labeled AcLDL or oxidized LDL (OxLDL). The uptake of DiI-AcLDL was similar in PPAR–/– and PPAR+/+ macrophages with the exception of the highest concentration of AcLDL (Figure 3A). In contrast, PPAR–/– macrophages had increased uptake of DiI-OxLDL (by 46.7%, 64.1%, and 93.3% at doses of 5, 10, and 20 µg/mL, respectively) compared with PPAR+/+ macrophages (Figure 3B).
|
To examine whether macrophage PPAR deficiency had an impact on cholesterol efflux, we used 2 alternative approaches: efflux of radiolabeled cholesterol and bulk changes in cellular cholesterol content. PPAR–/– macrophages had slightly (12%, P<0.05) lower levels of HDL-mediated cholesterol efflux than PPAR+/+macrophages (Figure 3C). Similarly, wild-type macrophages treated with WY14643 and HDL for 24 and 48 hours had a significantly greater decrease (18.2% and 14.7%, respectively) in total cholesterol mass than cells treated with HDL only (Figure 3D). Although there appeared to be a trend for a decrease (12.6% and 9.9%) in cholesterol mass in PPAR–/– macrophages, this difference was not statistically significant (Figure 3D). Thus, PPAR-deficient macrophages had increased OxLDL uptake and decreased levels of cholesterol efflux compared with wild-type cells.
PPAR Deficiency Affects CD36, SR-BI, and Inflammatory Response Genes
To evaluate the impact of macrophage PPAR on genes implicated in cholesterol homeostasis, peritoneal macrophages from wild-type and PPAR–/– mice were treated with the PPAR agonist WY14643 (2 µmol/L) for 24 hours. Quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that the agonist upregulates CD36 in a PPAR-independent manner (Figure 4A). Interestingly, WY14643 treatment increased the SR-BI gene expression levels (Figure 4B) and SR-BI protein content (19%) in PPAR+/+ macrophages but not in PPAR–/– macrophages (Figure 4C and 4D). In contrast, LPS treatment significantly decreased SR-BI gene expression (data not shown) and protein levels in both types of cells (41% and 31%) compared with untreated cells (Figure 4C and 4D). Similar effects on macrophage SR-B1 expression were seen with another PPAR agonist, GW7647 (Figure 5A). Wild-type macrophages treated with this ligand had a trend for an increase in ABCA1 and ABCG1 gene expression (Figure 5B and 5C), but only ABCG4 gene expression was statistically significant compared with PPAR–/– macrophages (Figure 5D). In addition, WY14643 treatment did not dramatically change ABCA1 protein expression in either type of macrophage, but wild-type cells had higher levels of ABCA1 protein content than PPAR–/– macrophages (Figure 5E and 5F).
|
|
Next, to examine whether macrophage PPAR has an impact on the inflammatory response, wild-type and PPAR–/– peritoneal macrophages were stimulated with LPS (50 ng/mL) for 5 hours, and the expression pattern of inflammatory genes was analyzed by real-time PCR. Compared with wild-type cells, PPAR–/– macrophages had significantly increased mRNA expression levels of inflammatory genes including COX-2, IL1ß, IL6, Gro1, MCP-1, and p65 (Figure 6A through 6F). Thus, macrophage PPAR deficiency eliminates PPAR-mediated SR-BI activation, suppresses ABCA1 protein production, and accelerates LPS-induced inflammatory responses.
|
SR-BI and Nuclear Factor-B Expression Levels in Atherosclerotic Lesions
Finally, to examine whether the expression of SR-BI and nuclear factor (NF)-B genes are compromised in vivo in atherosclerotic lesions, we used an LCM approach as reported by Trogan et al.29 Using serial sections of the proximal aorta stained with a specific anti-macrophage antibody (Figure 7A) as a guide, macrophage-rich atherosclerotic lesions were collected by LCM (Figure 7B through 7D). Real-time PCR analysis of amplified RNA samples indicated that PPAR–/– cells in atherosclerotic lesions had no changes in CD36, SR-A, or ABCA1 gene expression levels (data not shown) but had diminished expression of SR-BI (Figure 7E) and increased expression of p65 (Figure 7F) genes compared with control lesions with wild-type macrophages. Thus, mice reconstituted with PPAR–/– macrophages had lower levels of SR-BI and elevated levels of NF-B mRNA expression in atherosclerotic lesions in vivo.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the present study, the role of macrophage PPAR
expression in atherogenesis was examined in male and female LDLR–/– mice reconstituted with PPAR–/– or PPAR+/+ macrophages. Both male and female PPAR–/–LDLR–/– mice had significantly increased (44% and 46%, respectively) atherosclerotic lesions in the distal aorta compared with controls. Male PPAR–/–LDLR–/– mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPAR+/+LDLR–/– mice. The increase in atherosclerosis in these mice occurred in the absence of significant differences in serum lipids or lipoprotein profiles compared with controls. Thus, local macrophage PPAR expression in the artery wall has protective antiatherogenic effects in vivo.
The lack of a significant difference in the proximal aortas of the female mice may be the result of a sexual dimorphism specific for PPAR–/–.30 Female PPAR–/– mice are prone to develop obesity and have increased plasma triglycerides, whereas PPAR-null males have hepatic accumulation of triglycerides and higher serum cholesterol and apoB levels.30,31 Therefore, gender differences in atherosclerosis susceptibility may have contributed to the lack of difference in atherosclerosis in the proximal aortas of the females,32,33 although the mechanism for this sexual dimorphism remains unclear. Alternatively, accelerated formation of complicated lesions in the females with a low ratio of macrophage area to oil red O–staining area in the proximal aorta of female recipients may have obscured the effect of macrophage PPAR expression on lesion area.
Previous investigations of the role of PPAR in atherosclerosis in apoE–/– mice that used PPAR agonists and gene knockouts have yielded conflicting results. Mice treated with PPAR agonists had severe hypercholesterolemia and accelerated atherosclerosis12 but reduced cholesterol content in the descending aortas.34 In contrast, Tordjman et al11 reported that systemic PPAR deficiency in apoE–/– mice decreased insulin resistance, blood pressure, and atherosclerosis compared with apoE–/– mice. Recently, Bernal-Mizrachi et al35 identified PPAR expression in the liver as a key mediator of insulin resistance and hypertension in mice. Collectively, these results indicate that the systemic absence of PPAR does not produce the opposite phenotype of fibrate-mediated activation of PPAR and that the role of PPAR in atherosclerosis is complex.
Macrophage transformation into foam cells can be viewed as an imbalance between cholesterol influx attributed mainly to scavenger receptors type A and CD36 and to lipid efflux mediated by membrane transporters ABCA1, ABCG1, and SR-BI. Chinetti and coworkers36 have demonstrated that PPAR agonists induce cholesterol removal from human macrophage-derived foam cells through stimulation of the ABCA-1 pathway. In our studies, we present evidence that PPAR-null macrophages have increased CD36 gene expression levels and uptake of OxLDL. Although the CD36 receptor promoter does not have PPAR-response elements, PPAR can regulate the CD36 receptor gene via induction of other transcriptional factors such as Sp-1.37 Febbraio and coworkers38 demonstrated that CD36 expression by macrophages significantly accelerates atherosclerosis. Thus, activation of scavenger receptor CD36 expression in PPAR-deficient mice may have proatherogenic effects.
The present results indicate that PPAR deficiency suppresses cholesterol efflux in macrophages. Although the impact of PPAR on cholesterol efflux was relatively low (12% to 18%), this difference was statistically significant and consistently reproducible. Therefore, we believe that over time, differences of this magnitude may significantly impact macrophage cholesterol homeostasis. Treatment of macrophages cholesterol loaded in vivo with a PPAR agonist has been reported to reduce ABCA1 protein levels.13 Although PPAR activation in the present study did not change ABCA1 gene and protein expression levels in the absence of cholesterol loading, wild-type macrophages had significantly higher levels of ABCA1 protein content than PPAR–/– cells, consistent with an important role for PPAR in modulating ABCA1 posttranscriptionally. Given that Neufeld and coworkers39 have shown a role for ABCA1 in mobilizing lysosomal cholesterol, this may represent an important effect of PPAR on macrophage cellular cholesterol homeostasis. Interestingly, PPAR activation stimulated SR-BI gene and protein expression, and ABCG4 mRNA levels in wild-type but not in PPAR–/– macrophages. The impact of increased ABCG4 expression on cholesterol efflux is uncertain, because studies have shown that increased expression of ABCG4 promotes efflux,40 but elimination of ABCG4 gene expression does not appear to impact cholesterol efflux.41 The present data on SR-BI are consistent with the results of recent studies indicating that PPAR specifically induces gene activity.42 SR-BI mediates the efflux of cholesterol from macrophages to HDL and facilitates the selective uptake of cholesterol esters from lipoproteins into liver.43 Ji and coworkers44 demonstrated that macrophage-derived foam cells in atherosclerotic lesions of apoE–/– mice express abundant SR-BI. PPAR agonists upregulated SR-BI protein levels in human and murine macrophages,19,45 and SR-BI protein level is significantly increased in atherosclerotic lesions of apoE-null mice treated with a PPAR ligand.19 In contrast, mice lacking SR-BI exclusively in bone marrow–derived cells developed accelerated atherosclerosis compared with wild-type mice.46,47 In addition, we used the LCM approach to demonstrate directly that the SR-BI gene is expressed at higher levels in atherosclerotic lesions of mice reconstituted with wild-type macrophages than in mice transplanted with PPAR–/– marrow. Taken together, these results indicate that PPAR-mediated SR-BI activation may play an important protective role in atherosclerosis.
Previous studies demonstrated that PPAR null mice display a prolonged response to inflammatory stimuli48 and acquire a state of redox imbalance.49 PPAR agonists stimulate reactive oxygen species production50 and lipolysis with the generation of endogenous PPAR ligands51 that can further activate PPAR and subsequently limit inflammation.52 The agonists also inhibited the expression of LPS-mediated inflammatory cytokines (interleukin [IL]-1ß, IL6, IL8, and tumor necrosis factor-) by antagonizing the constitutively active NF-B.49,53 Here, we used a genetic approach to demonstrate that peritoneal macrophages execute these antiinflammatory effects. Deficiency of the PPAR gene in macrophages significantly accelerated LPS-mediated activation of the inflammatory response genes (such as COX-2, IL-1ß, IL-6, MCP-1, and p65). Moreover, macrophage-derived foam cells in atherosclerotic lesions of mice reconstituted with PPAR-null macrophages have increased expression levels of the NF-B transcription factor subunit p65 than control lesions reconstituted with wild-type cells. Although not definitive in the absence of protein data, these results are consistent with increased expression of NF-B–regulated genes. The results also suggest that macrophage PPAR deficiency promotes a proinflammatory phenotype, which has increased sensitivity to oxidative stress. Thus, macrophage PPAR expression suppresses the NF-B pathway, inducing protective and antiatherogenic effects in vivo within atherosclerotic lesions.
In summary, we demonstrate here that macrophage PPAR expression plays an important protective role in atherosclerotic lesion formation in vivo. Macrophage PPAR deficiency significantly increases uptake of oxidized LDL, eliminates the PPAR-mediated SR-BI activation, diminishes ABCA1 protein content, and accelerates the response to inflammatory stimuli. These results support the concept that local PPAR expression in the artery wall coordinates macrophage cholesterol homeostasis and inflammatory activity, thereby mediating protective antiatherogenic effects.
|
Acknowledgments |
|---|
The authors are grateful to Youmin Zhang for excellent technical expertise.
Sources of Funding
This work was supported by National Institutes of Health grants HL65405, American Heart Association grant 0555323B, and HL57986, HL65709, and DK59637-01 (Lipid, Lipoprotein and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Centers). Dr Babaev was supported by an American Heart Association grant.
Disclosures
None.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Fruchart JC, Staels B, Duriez P. PPARS, metabolic disease and atherosclerosis. Pharm Res. 2001; 44: 345–352.[CrossRef]
2. Mandard S, Muller M, Kersten S. Peroxisome proliferator-activated receptor alpha target genes. Cell Mol Life Sci. 2004; 61: 393–416.[CrossRef][Medline] [Order article via Infotrieve]
3. Linton MF, Fazio S. Re-emergence of fibrates in the management of dyslipidemia and cardiovascular risk. Curr Atheroscler Rep. 2000; 2: 29–35.[Medline] [Order article via Infotrieve]
4. Ericsson CG, Nilsson J, Grip L, Svane B, Hamsten A. Effect of bezafibrate treatment over five years on coronary plaques causing 20% to 50% diameter narrowing (the Bezafibrate Coronary Atherosclerosis Intervention Trial [BECAIT]). Am J Cardiol. 1997; 80: 1125–1129.[CrossRef][Medline] [Order article via Infotrieve]
5. Ruotolo G, Ericsson CG, Tettamanti C, Karpe F, Grip L, Svane B, Nilsson J, de Faire U, Hamsten A. Treatment effects on serum lipoprotein lipids, apolipoproteins and low density lipoprotein particle size and relationships of lipoprotein variables to progression of coronary artery disease in the Bezafibrate Coronary Atherosclerosis Intervention Trial (BECAIT). J Am Coll Cardiol. 1998; 32: 1648–1656.
6. Frick MH, Elo O, Haapa K, Heinonen OP, Heinsalmi P, Helo P, Huttunen JK, Kaitaniemi P, Koskinen P, Manninen V, Maenpaa H, Malkonen M, Norola S, Pasternak A, Pikkarainen J, Romo M, Sjoblom T, Nikkila EA. Helsinki Heart Study: primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia: safety of treatment, changes in risk factors, and incidence of coronary heart disease. N Engl J Med. 1987; 317: 1237–1245.[Abstract]
7. Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH, Linares E, Schaefer EJ, Schectman G, Wilt TJ, Wittes J; Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. N Engl J Med. 1999; 341: 410–418.
8. Keech A, Simes RJ, Barter P, Best J, Scott R, Taskinen MR, Forder P, Pillai A, Davis T, Glasziou P, Drury P, Kesäniemi YA, Sullivan D, Hunt D, Colman P, d’Emden M, Whiting M, Ehnholm C, Laakso M; the FIELD Study Investigators. Effects of long-term fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial [published corrections appear in Lancet. 2006;368:1415 and Lancet. 2006;368:1420]. Lancet. 2005; 366: 1849–1861.[CrossRef][Medline] [Order article via Infotrieve]
9. Barbier O, Torra IP, Duguay Y, Blanquart C, Fruchart JC, Glineur C, Staels B. Pleiotropic actions of peroxisome proliferator-activated receptors in lipid metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol. 2002; 22: 717–726.
10. Flavell DM, Jamshidi Y, Hawe E, Pineda Torra I, Taskinen MR, Frick MH, Nieminen MS, Kesaniemi YA, Pasternack A, Staels B, Miller G, Humphries SE, Talmud PJ, Syvanne M. Peroxisome proliferator-activated receptor alpha gene variants influence progression of coronary atherosclerosis and risk of coronary artery disease. Circulation. 2002; 105: 1440–1445.
11. Tordjman K, Bernal-Mizrachi C, Zemany L, Weng S, Feng C, Zhang F, Leone TC, Coleman T, Kelly DP, Semenkovich CF. PPARalpha deficiency reduces insulin resistance and atherosclerosis in apoE-null mice. J Clin Invest. 2001; 107: 1025–1034.[Medline] [Order article via Infotrieve]
12. Fu T, Kozarsky KF, Borensztajn J. Overexpression of SR-BI by adenoviral vector reverses the fibrate-induced hypercholesterolemia of apolipoprotein E-deficient mice. J Biol Chem. 2003; 278: 52559–52563.
13. Li AC, Binder CJ, Gutierrez A, Brown KK, Plotkin CR, Pattison JW, Valledor AF, Davis RA, Willson TM, Witztum JL, Palinski W, Glass CK. Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARalpha, beta/delta, and gamma. J Clin Invest. 2004; 114: 1564–1576.[CrossRef][Medline] [Order article via Infotrieve]
14. Hennuyer N, Tailleux A, Torpier G, Mezdour H, Fruchart J-C, Staels B, Fievet C. PPAR{alpha}, but not PPAR{gamma}, activators decrease macrophage-laden atherosclerotic lesions in a nondiabetic mouse model of mixed dyslipidemia. Arterioscler Thromb Vasc Biol. 2005; 25: 1897–1902.
15. Duez H, Chao Y-S, Hernandez M, Torpier G, Poulain P, Mundt S, Mallat Z, Teissier E, Burton CA, Tedgui A, Fruchart J-C, Fievet C, Wright SD, Staels B. Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice. J Biol Chem. 2002; 277: 48051–48057.
16. Chinetti G, Lestavel S, Fruchart J-C, Clavey V, Staels B. Peroxisome proliferator-activated receptor {alpha} reduces cholesterol esterification in macrophages. Circ Res. 2003; 92: 212–217.
17. Inoue I, Shino K, Noji S, Awata T, Katayama S. Expression of peroxisome proliferator-activated receptor alpha (PPAR alpha) in primary cultures of human vascular endothelial cells. Biochem Biophys Res Com. 1998; 246: 370–374.[CrossRef][Medline] [Order article via Infotrieve]
18. Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart J-C. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation. 1998; 98: 2088–2093.
19. Chinetti G, Gbaguidi FG, Griglio S, Mallat Z, Antonucci M, Poulain P, Chapman J, Fruchart JC, Tedgui A, Najib-Fruchart J, Staels B. CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors. Circulation. 2000; 101: 2411–2417.
20. Lee SS, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, Fernandez-Salguero PM, Westphal H, Gonzalez FJ. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol. 1995; 15: 3012–3022.[Abstract]
21. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993; 92: 883–893.[Medline] [Order article via Infotrieve]
22. Linton MF, Babaev VR, Gleaves LA, Fazio S. A direct role for the macrophage low density lipoprotein receptor in atherosclerotic lesion formation. J Biol Chem. 1999; 274: 19204–19210.
23. 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.
24. Babaev VR, Patel MB, Semenkovich CF, Fazio S, Linton MF. Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in LDL receptor deficient mice. J Biol Chem. 2000; 275: 26293–26299.
25. Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987; 68: 231–240.[CrossRef][Medline] [Order article via Infotrieve]
26. Chawla A, Barak Y, Nagy L, Liao D, Tontonoz P, Evans RM. PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat Med. 2001; 7: 48–52.[CrossRef][Medline] [Order article via Infotrieve]
27. Klansek JJ, Yancey P, St Clair RW, Fischer RT, Johnson WJ, Glick JM. Cholesterol quantitation by GLC: artifactual formation of short-chain steryl esters. J Lipid Res. 1995; 36: 2261–2266.[Abstract]
28. Babaev VR, Yancey PG, Ryzhov SV, Kon V, Breyer MD, Magnuson MA, Fazio S, Linton MF. Conditional knockout of macrophage PPARgamma increases atherosclerosis in C57BL/6 and low-density lipoprotein receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2005; 25: 1647–1653.
29. Trogan E, Choudhury RP, Dansky HM, Rong JX, Breslow JL, Fisher EA. Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 2002; 99: 2234–2239.
30. Vu-Dac N, Schoonjans K, Kosykh V, Dallongeville J, Fruchart JC, Staels B, Auwerx J. Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor. J Clin Invest. 1995; 96: 741–750.[Medline] [Order article via Infotrieve]
31. Linden D, Alsterholm M, Wennbo H, Oscarsson J. PPARalpha deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins. J Lipid Res. 2001; 42: 1831–1840.
32. Paigen B, Holmes PA, Mitchell D, Albee D. Comparison of atherosclerotic lesions and HDL-lipid levels in male, female, and testosterone-treated female mice from strains C57BL/6, BALB/c, and C3H. Atherosclerosis. 1987; 64: 215–221.[CrossRef][Medline] [Order article via Infotrieve]
33. Purcell-Huynh DA, Farese RV Jr, Johnson DF, Flynn LM, Pierotti V, Newland DL, Linton MF, Sanan DA, Young SG. Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high-fat diet. J Clin Invest. 1995; 95: 2246–2257.[Medline] [Order article via Infotrieve]
34. Duez H, Fruchart JC, Staels B. PPARS in inflammation, atherosclerosis and thrombosis. J Cardiovasc Risk. 2001; 8: 187–194.[CrossRef][Medline] [Order article via Infotrieve]
35. Bernal-Mizrachi C, Weng S, Feng C, Finck BN, Knutsen RH, Leone TC, Coleman T, Mecham RP, Kelly DP, Semenkovich CF. Dexamethasone induction of hypertension and diabetes is PPAR-alpha dependent in LDL receptor-null mice. Nat Med. 2003; 9: 1069–1075.[CrossRef][Medline] [Order article via Infotrieve]
36. Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, Teissier E, Minnich A, Jaye M, Duverger N, Brewer HB, Fruchart JC, Clavey V, Staels B. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med. 2001; 7: 53–58.[CrossRef][Medline] [Order article via Infotrieve]
37. Sato O, Kuriki C, Fukui Y, Motojima K. Dual promoter structure of mouse and human fatty acid translocase/cd36 genes and unique transcriptional activation by peroxisome proliferator-activated receptor alpha and gamma ligands. J Biol Chem. 2002; 277: 15703–15711.
38. Febbraio M, Podrez EA, Smith JD, Hajjar DP, Hazen SL, Hoff HF, Sharma K, Silverstein R. Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice. J Clin Invest. 2000; 105: 1049–1056.[Medline] [Order article via Infotrieve]
39. Neufeld EB, Stonik JA, Demosky SJ Jr, Knapper CL, Combs CA, Cooney A, Comly M, Dwyer N, Blanchette-Mackie J, Remaley AT, Santamarina-Fojo S, Brewer HB Jr. The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease. J Biol Chem. 2004; 279: 15571–15578.
40. Wang N, Lan D, Chen W, Matsuura F, Tall AR. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci U S A. 2004; 101: 9774–9779.
41. Wang N, Ranalletta M, Matsuura F, Peng F, Tall AR. LXR-induced redistribution of ABCG1 to plasma membrane in macrophages enhances cholesterol mass efflux to HDL. Arterioscler Thromb Vasc Biol. 2006; 26: 1310–1316.
42. Lopeza D, McLean MP. Activation of the rat scavenger receptor class B type I gene by PPAR[alpha]. Mol Cell Endocrinol. 2006; 251: 67–77.[CrossRef][Medline] [Order article via Infotrieve]
43. Yancey PG, Asztalos BF, Stettler N, Piccoli D, Williams DL, Connelly MA, Rothblat GH. SR-BI- and ABCA1-mediated cholesterol efflux to serum from patients with Alagille syndrome. J Lipid Res. 2004; 45: 1724–1732.
44. Ji Y, Jian B, Wang N, Sun Y, Moya ML, Phillips MC, Rothblat GH, Swaney JB, Tall AR. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux. J Biol Chem. 1997; 272: 20982–20985.
45. Chinetti-Gbaguidi G, Fruchart JC, Staels B. Pleiotropic effects of fibrates. Curr Atheroscler Rep. 2005; 7: 396–401.[Medline] [Order article via Infotrieve]
46. Covey SD, Krieger M, Wang W, Penman M, Trigatti BL. Scavenger receptor class B type I-mediated protection against atherosclerosis in LDL receptor-negative mice involves its expression in bone marrow-derived cells. Arterioscler Thromb Vasc Biol. 2003; 23: 1589–1594.
47. Zhang W, Yancey PG, Su YR, Babaev VR, Zhang Y, Fazio S, Linton MF. Inactivation of macrophage scavenger receptor class B type I promotes atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation. 2003; 108: 2258–2263.
48. Devchand PR, Keller H, Peters JM, Vazquez M, Gonzalez FJ, Wahli W. The PPARalpha-leukotriene B4 pathway to inflammation control. Nature. 1996; 384: 39–43.[CrossRef][Medline] [Order article via Infotrieve]
49. Poynter ME, Daynes RA. Peroxisome proliferator-activated receptor alpha activation modulates cellular redox status, represses nuclear factor-kappaB signaling, and reduces inflammatory cytokine production in aging. J Biol Chem. 1998; 273: 32833–32841.
50. Teissier E, Nohara A, Chinetti G, Paumelle R, Cariou B, Fruchart J-C, Brandes RP, Shah A, Staels B. Peroxisome proliferator-activated receptor {alpha} induces NADPH oxidase activity in macrophages, leading to the generation of LDL with PPAR-{alpha} activation properties. Circ Res. 2004; 95: 1174–1182.
51. Ziouzenkova O, Perrey S, Asatryan L, Hwang J, MacNaul KL, Moller DE, Rader DJ, Sevanian A, Zechner R, Hoefler G, Plutzky J. Lipolysis of triglyceride-rich lipoproteins generates PPAR ligands: evidence for an antiinflammatory role for lipoprotein lipase. Proc Natl Acad Sci U S A. 2003; 100: 2730–2735.
52. Devchand PR, Ziouzenkova O, Plutzky J. Oxidative stress and peroxisome proliferator-activated receptors: reversing the curse? Circ Res. 2004; 95: 1137–1139.
53. Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart J-C, Tedgui A, Haegeman G, Staels B. Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappa B and AP-1. J Biol Chem. 1999; 274: 32048–32054.
 |
|
 |
|
  L. Piqueras, M. J. Sanz, M. Perretti, E. Morcillo, L. Norling, J. A. Mitchell, Y. Li, and D. Bishop-Bailey Activation of PPAR{beta}/{delta} inhibits leukocyte recruitment, cell adhesion molecule expression, and chemokine release J. Leukoc. Biol., July 1, 2009; 86(1): 115 - 122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Rigamonti, G. Chinetti-Gbaguidi, and B. Staels Regulation of Macrophage Functions by PPAR-{alpha}, PPAR-{gamma}, and LXRs in Mice and Men Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1050 - 1059. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||