PPAR Regulates Multiple Proinflammatory Pathways To

PPAR␦ regulates multiple proinflammatory pathways to suppress atherosclerosis

Grant D. Barish*†, Annette R. Atkins*, Michael Downes*, Peter Olson*‡, Ling-Wa Chong*, Mike Nelson*, Yuhua Zou*, Hoosang Hwang§, Heonjoong Kang§, Linda Curtiss¶, Ronald M. Evans*ʈ, and Chih-Hao Leeʈ**

*Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037; †Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Francisco, CA 94121; ‡Department of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093; §Marine Biotechnology Laboratory, Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University NS-80, Seoul 151-747, Korea; ¶Department of Immunology and Vascular Biology, Scripps Research Institute, La Jolla, CA 92037; and **Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115

Contributed by Ronald M. Evans, December 23, 2007 (sent for review September 5, 2007) Lipid homeostasis and inflammation are key determinants in addition, PPAR␦ has been implicated in keratinocyte homeosta- atherogenesis, exemplified by the requirement of lipid-laden, sis and hepatic lipoprotein production (25–27). foam cell macrophages for atherosclerotic lesion formation. Al- Based on their functions in lipid metabolism and inflamma- though the nuclear receptor PPAR␦ has been implicated in both tion, the potential for PPARs to modulate atherosclerosis has systemic lipid metabolism and macrophage inflammation, its role been explored. Several studies have demonstrated that PPAR␥ as a therapeutic target in vascular disease is unclear. We show here agonists reduce vascular lesion size, in part, by activating the that orally active PPAR␦ agonists significantly reduce atheroscle- LXR␣-ABCA1 pathway and directly regulating ABCG1 in the ؊ ؊ rosis in apoE / mice. Metabolic and gene expression studies macrophage to promote cholesterol efflux (28–30). A high- reveal that PPAR␦ attenuates lesion progression through its HDL- affinity PPAR␦ agonist GW501516 has been shown to up- raising effect and anti-inflammatory activity within the vessel regulate ABCA1 expression in human monocytic cell lines and wall, where it suppresses chemoattractant signaling by down- increase high-density lipoprotein cholesterol (HDL-c) in mon- ␦ regulation of chemokines. Activation of PPAR also induces the keys (31), suggesting that PPAR␦ may suppress atherogenesis expression of regulator of G protein signaling (RGS) genes, which through a similar mechanism. However, our previous study using are implicated in blocking the signal transduction of chemokine both loss- and gain-of-function approaches and work by others ␦ receptors. Consistent with this, PPAR ligands repress monocyte indicates that PPAR␦ does not directly regulate cholesterol transmigration and macrophage inflammatory responses elicited ␦ efflux in the mouse macrophage (30, 32). Instead, it regulates the by atherogenic cytokines. These results reveal that PPAR antag- metabolism of very-low-density lipoprotein-derived fatty acid onizes multiple proinflammatory pathways and suggest PPAR␦- and is capable of down-regulating inflammatory mediators selective drugs as candidate therapeutics for atherosclerosis. including IL-1␤ and MCP-1 (32, 33). Recently, two independent studies examined the effect of a less characterized PPAR␦ inflammation ͉ ligand ͉ mouse ͉ nuclear ͉ receptor MEDICAL SCIENCES agonist GW0742 on lesion progression in fat- and cholesterol- supplemented LdlrϪ/Ϫ mice and produced divergent results (30, xtensive research implicates inflammation associated with 34). In the first study, GW0742 treatment did not affect the total Elipid dysregulation in the pathogenesis of atherosclerosis (1, lesion area after 14 weeks of drug treatment. In the second study, 2). A network of proinflammatory cytokines, including inter- ␤ ␤ ␣ ␣ ␥ GW0742 reduced the lesion size after 10 weeks of treatment but leukin-1 (IL-1 ), tumor necrosis factor (TNF ), and IFN , only through a more aggressive dosing regimen. Neither study, trigger chemokine production, leukocyte recruitment, smooth however, detected the increase in HDL-c associated with muscle cell migration, and ultimately, plaque rupture. The GW501516 treatment. crucial role for chemokines in this process is exemplified by Because it remains unclear whether PPAR␦ drugs could studies demonstrating that mice lacking monocyte chemoattrac- modulate atherosclerosis, we examined the effect of GW501516 tant protein-1 (MCP-1/CCL2) or its receptor CCR2 are highly Ϫ Ϫ (GW) on lesion development in apoE / mice. Low doses of the protected against atherosclerosis (3–6). Thus, therapeutic ma- PPAR␦ agonist GW501516 significantly reduced atherosclerotic nipulation to improve serum lipid profiles or attenuate inflam- lesions and increased HDL-c, although they had no effect on mation is expected to control the progression of this disease. Peroxisome proliferator-activated receptors (PPARs) are nu- total cholesterol levels. Expression profiling of the aortas of clear receptors activated by dietary fats and have critical func- treated mice suggested that multiple chemokine-mediated cell ␣ migration pathways are down-regulated by ligand treatment. tions in lipid homeostasis (7, 8). PPAR is the target of the ␦ fibrate class of lipid-lowering drugs (9–11). It is most highly Consistent with this observation, activation of PPAR represses expressed in the liver where it regulates fatty acid ␤-oxidation (8, the expression of chemoattractants MCP-1, MCP-3, and MCP-5 ␤ ␥ 12). PPAR␥ is essential for adipocyte differentiation and fat induced by IL-1 and IFN in cultured macrophages. In addi- storage (13–16). It is also the molecular target of the thiazo- tion, monocytes pretreated with GW501516 exhibit reduced lidinedione (TZD) class of insulin-sensitizing drugs (17, 18). transendothelial migration. These results provide molecular ␦ PPAR␦ (also known as PPAR␤) has the broadest expression of targets through which PPAR suppresses atherogenic inflam- the PPAR isotypes, and its biological function has recently been examined through genetic manipulation (19–21). Restricted Author contributions: G.D.B., R.M.E., and C.-H.L. designed research; G.D.B., A.R.A., M.D., activation of this receptor in either muscle or adipose tissue P.O., L.-W.C., M.N., Y.Z., and C.-H.L. performed research; H.H. and H.K. contributed new results in a lean phenotype because of increased fatty acid reagents/analytic tools; G.D.B., A.R.A., M.D., P.O., L.C., and C.-H.L. analyzed data; and ␤-oxidation (22, 23). Long-term treatment with a PPAR␦ in- G.D.B., R.M.E., and C.-H.L. wrote the paper. vestigational ligand GW501516 causes dramatic weight loss The authors declare no conflict of interest. accompanied by improvements in lipoprotein profiles (23). ʈTo whom correspondence may be addressed. E-mail: [email protected] or clee@hsph. Recently, we have shown that ligand treatment reduces hyper- harvard.edu. glycemia and improves insulin sensitivity in db/db mice (24). In © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711875105 PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4271–4276 Downloaded by guest on September 24, 2021 Fig. 2. PPAR␦ increases HDL-c in apoEϪ/Ϫ mice. (A) GW does not affect weight gain. (B–D) Similar levels of serum triglyceride, free fatty acid (FFA), and cholesterol are observed between vehicle- and GW-treated groups. C, chow diet; HF, high-fat diet; HFϩGW, high-fat diet with GW treatment. (E)An Ϫ Ϫ increase in HDL cholesterol occurs in GW-treated apoE / mice. *, P Ͻ 0.05.

reduced aortic valve lesions by 20% (Fig. 1E; control, 504,904.2 Ϯ 33,357.3; GW, 403,632.2 Ϯ 2,5261.1 ␮m2, P ϭ 0.016). Fig. 1. PPAR␦ activation protects against atherosclerosis. (A and B) Repre- sentative oil red O-stained sections of aortic valves from vehicle (A)or Similarly, GW decreased the number of fatty streaks throughout GW501516 (GW)-treated (B) apoEϪ/Ϫ mice. Ten-week-old male apoEϪ/Ϫ mice the aorta (Fig. 1 C and D) with a 30% reduction in total lesion on an atherogenic diet were gavaged daily with either vehicle or GW, a area (control, 9.25 Ϯ 0.55; GW, 6.53 Ϯ 0.46%, P ϭ 0.002) (Fig. high-afﬁnity PPAR␦ agonist, at 2 mg⅐kgϪ1⅐dayϪ1 for 8 weeks. (C and D) En face 1F). Serum GW501516 concentrations were Ϸ106 nM in drug- lesion area in representative vehicle (C) or GW-treated (D) aortas. (E and F) treated mice and undetectable in controls, as determined by ϭ Quantitative analysis of the lesion size in aortic valves (E) and aortas (F)(n liquid chromatography-tandem mass spectrometry (LC-MS/MS) 15). GW treatment signiﬁcantly reduced lesion sizes. , P Ͻ 0.05; , P Ͻ 0.005 * ** (Fig. 1G). The former concentrations of GW exceed its reported Mann–Whitney U test. (G) LC-MS/MS quantitation of GW drug levels in pooled ␦ serum from vehicle (Inset Left) versus drug-treated mice. Standard curve for EC50 for murine PPAR (20 nM) but are far below the EC50 for quantitation of GW levels (Inset Right). PPAR␣ (2.5 ␮M) or PPAR␥ (1 ␮M), indicating that specific activation of PPAR␦ inhibits lesion formation (31, 36). mation and substantiate PPAR␦-selective drugs as potential PPAR␦ Increases HDL-c in apoE؊/؊ Mice. To determine the systemic therapeutics to treat atherosclerosis. effects of ligand administration, metabolic parameters were measured. Both control and ligand-treated groups consumed an Results equal amount of food and gained similar weight during the PPAR␦ Inhibits Atherosclerotic Lesion Formation. A high-affinity ␦ experimental period (Fig. 2A and data not shown). Because PPAR agonist, GW501516 (GW), has been reported to in- long-term treatment of GW compound at a higher dose causes crease HDL-c and reduce circulating triglycerides in monkeys weight loss (23, 37), this regimen minimized the indirect effects and humans (31, 35). To determine whether PPAR␦ could resulting from weight differences. Interestingly, circulating levels modulate atherosclerotic lesion progression, we examined the Ϫ/Ϫ of glucose (control, 164.06 Ϯ 3.65; GW, 155.73 Ϯ 3.92 mg/dl), effect of GW on lesion development in apoE mice. Ten- Ϯ Ϯ week-old male apoEϪ/Ϫ mice were placed on a high-fat (HF) diet insulin (control, 0.71 0.08; GW, 0.75 0.13 ng/ml) and and treated with either vehicle or GW (n ϭ 15 for each group) triglycerides (Fig. 2B) were not elevated on this diet, nor were at2mg⅐kgϪ1⅐dayϪ1 for 8 weeks. Atherosclerotic lesions were they affected by GW treatment. GW also had no effect on levels subsequently determined by two methods: cross-sectional anal- of free fatty acid or cholesterol, which were increased signifi- ysis of the aortic valves and en face analysis of the aorta. cantly after HF challenge (Fig. 2 C and D). HDL-c, however, was Examination of the oil red O-stained area of the aortic valves lowered by HF and was restored by GW to levels similar to those revealed fewer lesions in GW-treated mice (Fig. 1 A and B). of apoEϪ/Ϫ mice on a normal chow diet (Fig. 2E). This HDL- Quantitative analyses further suggested that ligand treatment raising effect is consistent with results in monkeys but contrasts

4272 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711875105 Barish et al. Downloaded by guest on September 24, 2021 Table 1. PPAR␦ regulated genes identiﬁed by DNA array analysis Array* Q-PCR*

Inﬂammation Regulator of G protein signaling 3 1.5‡ (Mm.286753†) Regulator of G protein signaling 4 1.4 1.4 (Mm.54164) Regulator of G protein signaling 5 1.5 1.5 (Mm.20954) Tissue inhibitor of 1.4 1.4 metalloproteinase 3 (Mm.132958) Interferon-stimulated protein Ϫ1.5 Ϫ1.7 (Isg20, Mm.19019) CXCL7 (Mm.293614) Ϫ2.5 Ϫ1.8 CCL21␣ (Mm.22085) Ϫ1.4 Ϫ1.7 Resistin (Mm.1181) Ϫ1.4 Ϫ1.8 Chemokine-like factor superfamily Ϫ1.6 6 (Mm.28858) Lipid oxidation Carnitine palmitoyltransferase 1 1.8 1.9 (Mm.18522)

*Minus sign indicates down-regulation. †UniGene number. ‡Fold changes comparing ligand treated to the control group.

with previous studies in LdlrϪ/Ϫ mice that used the alternative PPAR␦ agonist GW0742 (30, 34).

Chemokine Signaling Pathways Are the Major Targets of PPAR␦ in Vivo. To understand how PPAR␦ modulates lesion progression, we isolated aortas from control and ligand-treated mice to compare the expression profiles at lesion sites by using DNA

array analysis followed by real-time PCR verification. Consistent MEDICAL SCIENCES with our previous study, no cholesterol efflux genes were Fig. 3. Real-time PCR analyses of potential PPAR␦ target genes in the aorta. affected by ligand treatment. At this low dose, carnitine palmi- ␦ ␦ Veriﬁcation of PPAR regulated genes identiﬁed by DNA array experiments toyltransferase 1 (CPT-1), a known PPAR target gene regu- and examination of additional atherosclerosis-related genes by using whole lating the rate-limiting step of fatty acid ␤-oxidation (22, 37), was aorta RNA from vehicle- or GW-treated apoEϪ/Ϫ mice. Results are presented as the only verifiable gene related to lipid homeostasis identified in mean (of three mice) Ϯ SEM. *, P Ͻ 0.05. the array (Table 1 and Fig. 3). The majority of the regulated genes are involved in inflammatory signaling pathways (Table 1 and Fig. 3). Changes include the down-regulation of IFN- regulatory mechanism may account for the reduced lesion area stimulated protein (Isg20), CXCL7, CCL21␣, resistin, and che- by GW, in addition to its HDL-raising effect. mokine-like factor superfamily 6 and up-regulation of regulator of G protein signaling (RGS) 4 and 5 and tissue inhibitor of PPAR␦ Suppresses Chemokine Expression in Vitro. The ability of GW metalloproteinase 3 (TIMP3). Among these, chemokines to down-regulate the expression of several chemokines CXCL7 and CCL21␣ have been shown to induce neutrophil and prompted us to investigate whether PPAR␦ does so by attenu- T lymphocyte recruitment, respectively (38–41), whereas RGSs ating signaling pathways initiated by proatherogenic cytokines. antagonize the signaling mediated by chemokine receptors, and Expression analysis showed that GW treatment significantly TIMP3 inhibits smooth muscle cell migration and maintains down-regulated MCP-1, MCP-3, and MCP-5 expression by lesion stability (42, 43). macrophages stimulated with IL-1␤, IFN␥, and phorbal 12- Ϫ Ϫ We also examined the expression of other factors critical for myristate 13-acetate (PMA). This was abolished in PPAR␦ / lesion development, which were either not included in the gene cells, indicating a receptor-dependent mechanism (Fig. 4 A–C). collection of the array or did not show differences by the analysis. Consistent with this, MCP-1 expression was reduced by 50% in Ϫ Ϫ Consistent with our previous results in cultured macrophages, primary macrophages derived from GW-treated apoE / mice real-time PCR demonstrated that GW suppressed the expression compared with vehicle controls (Fig. 4D). To determine whether of MCP-1 and IL-1␤ but not TNF␣ or MMP-9 (Fig. 3), whereas this regulation was of functional consequence, we examined the IFN␥ was undetectable (data not shown). Known PPAR␥ targets effect of GW on transmigration. Monocytic THP-1 cells were ABCA1 (29), CD36 (44), iNOS (45), and LXR␣ (29) (Fig. 3) preincubated with either vehicle or GW and subjected to an were not altered, nor was there a difference in the expression of MCP-1 gradient compartmented by an endothelial cell mono- PPAR␦ or PPAR␥ (data not shown). These data implicate layer. GW treatment significantly reduced the number of THP-1 PPAR␦ and PPAR␥ in distinct transcriptional programs to monocytes that migrated through the endothelial layer (Fig. 4E). ameliorate atherogenesis and suggest that chemokine-mediated Collectively, these results suggest that PPAR␦ can attenuate cell migration is a major target of PPAR␦ in the aorta. Because multiple inflammatory signaling pathways to inhibit cell migra- leukocyte recruitment plays a critical role in atherogenesis, this tion and lesion progression.

Barish et al. PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4273 Downloaded by guest on September 24, 2021 molecular mechanism underlying this observation remains to be determined, given that PPAR␦ does not regulate genes involved in cholesterol uptake or efflux in aortas. It is unlikely that the atheroprotective effects of PPAR␦ are contributed solely by its modest enhancement of HDL-c, however, and the anti- inflammatory aspects of PPAR␦ function may play a more dominant role. Consistent with this notion, our data suggest that the anti- inflammatory aspects of the PPAR␦ function may have an important role in inhibiting leukocyte recruitment and thus lesion progression by down-regulating the expression of chemokines, attenuating chemokine receptor signaling, and inhibiting matrix metalloproteinases (Fig. 3). Indeed, expression analysis demonstrates multitiered suppression of chemoattractant signaling by PPAR␦ (Fig. 4F). First, it down-regulates the expression of chemokines including MCP-1, CXCL7, and CCL21, which mediate the recruitment of macrophages, neutrophils, and T lymphocytes, respectively. In vitro studies suggest that PPAR␦ does so by suppressing inflammatory responses elicited by cytokines such as IL-1␤ and IFN␥ (Fig. 4 A–C). In addition, PPAR␦ may attenuate chemokine receptor signaling by the induction of RGS proteins, which interact with G␣ subunits to accelerate GTP hydrolysis and terminate G protein signals (42). Although the functions of these family members are not well defined, several of them, including RGS4, have been shown to antagonize chemokine receptor-mediated lymphocyte migration when overexpressed (46). RGS5, however, is a marker for arterial smooth muscle cells. Its expression is down-regulated in atherosclerotic plaques, whereas its up-regulation correlates with statin-induced atheroprotection in apoEϪ/Ϫ mice (47–49). In independent studies, we have identified RGS genes 1, 10, 16, and 18 as PPAR␦-regulated targets in the macrophage, suggesting modulation of G protein-coupled pathways as a common regulatory mechanism of this receptor (data not shown). Last, PPAR␦ may block cell migration through TIMP-3, which inhibits the activity of matrix metalloproteinases (MMPs), thereby main- taining integrity of the extracellular matrix. TIMP-3 can also inhibit TNF␣-converting enzyme (TACE), resulting in reduced TNF shedding, indicating that PPAR␦ may suppress TNF␣ signaling posttranslationally (50). Fig. 4. Activation of PPAR␦ attenuates chemoattractant signaling. (A–C) The basis for the discrepant effects of PPAR␦ ligands on Real-time PCR demonstrating ligand treatment suppresses chemokine expres- atherosclerosis observed in this versus two prior studies (30, 34), sion induced by IL-1␤, IFN␥, and PMA in a receptor-dependent manner. which used a less characterized PPAR␦ agonist GW0742, is Thioglycollate-elicited peritoneal macrophages were isolated from wild-type unclear. Several lines of evidence suggest that chemically distinct (wt) or PPAR␦Ϫ/Ϫ mice and stimulated with the indicated stimulant in the PPAR␦ agonists exhibit different biological activities. For in- presence or absence of ligand. (D) Reduced MCP-1 expression in peritoneal stance, at high doses, GW501516 causes weight loss, decreases Ϫ/Ϫ ␦ macrophages isolated from ligand treated apoE mice. (E) PPAR ligand serum TGs, and increases HDL-c, whereas GW0742 increased treatment inhibits monocyte transmigration. THP-1 monocytes pretreated with vehicle or GW were examined for their ability to migrate through an weight gain and has weak effects on TGs and no effects on endothelial cell monolayer along an MCP-1 gradient. (F) Schematic demon- HDL-c (34). We show that treatment with GW501516 at 2 Ϫ1 Ϫ1 strating that PPAR␦ inhibits inﬂammatory signaling through multiple down- mg⅐kg ⅐day significantly reduces lesions in 8 weeks, whereas Ϫ Ϫ stream effectors. We have previously shown that PPAR␦ regulates the expres- GW0742 is effective only at 60 mg⅐kg 1⅐day 1 for 10 weeks. In sion of the cytokine IL-1␤ and ligand-bound PPAR␦ releases the transcriptional the latter case, drug administration resulted in signs of severe repressor BCL-6 to repress the expression of chemokines such as MCP-1. In this liver enlargement and serum drug levels up to 21 ␮M, which is study, RGSs and TIMP-3 are shown to be up-regulated by PPAR␦ activation. 420- and 700-fold higher than the reported EC50 for mouse and RGSs block chemokine receptor signal transduction, whereas TIMP-3 inhibits human PPAR␦, respectively, raising questions about drug effi- ␣ Ͻ TNF shedding and cell migration. *, P 0.05. cacy and specificity in vivo. We have recently demonstrated that GW501516 increases insulin sensitivity in a receptor-dependent manner in obese mice dosed with a similar drug regimen (24). Discussion Ϸ ␦ Moreover, the 106 nM serum concentrations of GW501516 Significant efforts have focused on the role of PPAR in lipid detected with drug treatment in this study indicate that the homeostasis because of its ability to increase fatty acid oxidation. atheroprotection herein observed is very likely mediated by ␦ This study uncovers regulatory pathways for PPAR to limit PPAR␦. atherosclerosis by improving systemic lipid profiles and repress- Besides distinctions in the chemical properties and biological ing inflammation. HDL-c is believed to be protective against activities of PPAR␦ drug compounds, the mouse models and atherosclerosis. Synthetic ligands of PPAR␦ have been shown to diets used distinguish the studies to date. Hypercholesterolemia increase HDL-c in monkeys and rodents and thus may have is clearly essential for atherogenesis to proceed because there are therapeutic potential to treat this disease (31). We have also no animal models without it, and the degree of atherosclerosis observed increased HDL-c in GW-treated apoEϪ/Ϫ mice. The is related to the degree of hypercholesterolemia. However,

4274 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711875105 Barish et al. Downloaded by guest on September 24, 2021 inflammation is also a dominant factor promoting lesion forma- Metabolic Measurement. Serum and plasma were collected after6hoffasting. tion, and although likely proportional to hypercholesterolemia, Total cholesterol, triglyceride, and free fatty acids were determined by using it is influenced by many other factors. In the study by Li et al. enzymatic reactions (Thermo DMA; free fatty acid, Wako Chemicals). HDL (30), in which no atheroprotection for the PPAR␦ drug GW0742 cholesterol was determined by using a HDL cholesterol dextran sulfate- precipitating reagent set (Pointe Scientific). Blood glucose was measured was observed, it is possible that the extensive, 1.25% dietary through the tail tip by using the OneTouch (Lifescan) glucose-monitoring cholesterol content, versus 0.25% in Graham et al. (34) or 0.15% system. Insulin was measured by using RIA kits (Linco). Statistics were per- cholesterol in this study, produced a model in which the hyper- formed by using Student’s t test. Values are presented as means Ϯ SEM. lipidemic factor overshadowed other inflammatory components. Significance was established at P Ͻ 0.05. With the exception of patients with homozygous familial hypercholesterolemia, such hypercholesterolemia is not likely to occur Gene Expression Analysis. RNA was isolated by using TRIzol reagent (Invitro- in humans. gen). For DNA array analysis, aorta RNA samples from three mice of each Because the evidence is that the activity of PPAR␦ is pre- group were amplified and hybridized to GeneChip 430A 2.0 (Affymetrix). dominantly anti-inflammatory, human atherogenesis, typified by Target genes were determined by using both ANOVA (P Ͻ 0.05) and fold Ͼ more modest hypercholesterolemia, or murine models of athero- change ( 1.4-fold) with the assistance of the GeneSpring software (Silicon Genetics) and the Bullfrog program. The results were confirmed by real-time genesis in which inflammation is amplified, are predicted to be ␮ ␦ PCR. For real-time PCR, 1 g of RNA was DNase-treated and reverse- more amenable to therapeutic intervention by PPAR drugs. transcribed by using oligo(dT) (SuperScript II kit; Invitrogen) and then treated Together with its functions in fatty acid and HDL-c metabolism, with RNase. Samples were run in triplicate with SYBR Green (Applied Biosys- PPAR␦ represents an attractive therapeutic target for drug tems) and compared with levels of 36B4 as a control. development to treat atherosclerosis. Macrophage Studies. Resident macrophages were isolated from the perito- Materials and Methods neum of vehicle- or GW-treated apoEϪ/Ϫ mice before collecting their aortas to Animal Experiments. PPAR␦ knockout mice were generated as described in ref. analyze the expression of MCP-1 in vivo. For in vitro studies, peritoneal 19. Ten-week-old male apoEϪ/Ϫ mice under C57 background were purchased macrophages were isolated 3 days after thioglycollate challenge and cultured from The Jackson Laboratory (stock no. 002052) and challenged with an in RPMI, 10% FBS. Cells were treated with the indicated stimulant in the atherogenic diet (0.15% cholesterol and 21% fat, TD88137, Harlan Teklad) for presence or absence of ligand for 18 h before harvesting. Concentrations used 8 weeks. These animals were divided into two groups (15 mice per group); one were: GW501516, 0.1 ␮M; IL-1␤, 1 ng/ml; IFN␥, 100 units/ml; and PMA, 25 was given GW501516 at 2 mg⅐kgϪ1⅐dayϪ1 daily by gavage and the other was ng/ml. Cytokines and chemokines were purchased from BD Biosciences given vehicle control. GW501516 was dissolved in DMSO and suspended in (PharMingen). 0.5% carboxymethylcellulose. All animal procedures were approved by and For the transmigration assay, THP-1 cells (1 ϫ 105) were pretreated with carried out under the guidelines of the Institutional Animal Care and Use GW501516 overnight and added to a human umbilical vein endothelial cell Committee of the Salk Institute. monolayer covering a gelatin-coated membrane (8 ␮m, Transwell, Costar). Migration was induced by the addition of human monocyte chemoattractant Lesion Analysis. Mice were killed and perfused with 4% paraformaldehyde. protein-1 (MCP-1; 100 ng/ml) to the lower compartment. Migrated cells were Atherosclerotic lesions at aortic valves were identified by oil red O staining. determined after 30 min. The mean lesion area for each animal was determined by five sections taken every 40 ␮m. To examine lesions in the aorta, aortas were dissected out and ACKNOWLEDGMENTS. We thank Drs. J. Plutzky (Harvard Medical School), R. stained with Sudan IV. The extent of lesion formation is expressed as the Tangirala [University of California, Los Angeles (UCLA)], W. Hsueh (UCLA), C.

percentage of the total aortic surface area covered by lesions. Images were Glass [University of California at San Diego (UCSD)], and J. Witztum (UCSD) for MEDICAL SCIENCES taken by a video camera and lesion area was determined by using Adobe valuable comments, G. Bradshaw for help in lesion analyses, and S. Ganley and E. Ong for administrative assistance. This work was supported by National Photoshop and National Institutes of Health Scion Image software. Statistics Institutes of Health (NIH) National Research Service Award Grants T32 were performed by using the Mann–Whitney U test. CA009370, T32 HL007770, and F32 DK071478 (to G.D.B.); by CMG training grant (Department of Biology, UCSD) (P.O.), the Chapman Fellowship and the Liquid Chromatography-Tandem Mass Spectrometry. Solid-phase extraction on Molecular Neurobiology grant at the Salk Institute (P.O.), and the Achieve- Strata-X columns (Phenomenex) was used to prepare 100-␮l samples of pooled ment Rewards for College Students Foundation (P.O.); MarineBio 21 (Ministry serum containing 100 ng of internal standard (GW0742) for LC-MS/MS quan- of Maritime Affairs and Fisheries, Korea) (H.J.K.); and by the American Heart titation. The treatment drug and internal standard were subsequently re- Association and American Diabetes Association (C.H.L.) and the Howard Hughes Medical Institute, NIH Grant 5R37DK057978, UCSD Specialized Center solved on a 2 ϫ 50 mm Synergi Polar-RP column by using an acetonitrile/0.1% for Research in Molecular Medicine and Atherosclerosis Grant 5P50HL56989, formic acid gradient and detected by selective reaction monitoring (452/394, and the NIH Nuclear Receptor Signaling Atlas orphan receptor program Grant GW1516; 470/412, and GW0742) on a Thermo LTQ-XL mass spectrometer. A U19DK62434-01. R.M.E. is an investigator of the Howard Hughes Medical standard curve was constructed by using drug-spiked mouse serum to facili- Institute at the Salk Institute for Biological Studies and March of Dimes Chair tate quantitation. in Molecular and Developmental Biology.

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