HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 47, 44258-44265, November 23, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/47/44258    most recent M108482200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vosper, H. Articles by Palmer, C. N. A. Search for Related Content PubMed PubMed Citation Articles by Vosper, H. Articles by Palmer, C. N. A. Social Bookmarking                      What's this?

The Peroxisome Proliferator-activated Receptor  Promotes Lipid Accumulation in Human Macrophages^*

Helen Vosper, Lisa Patel^§, Tracey L. Graham, Guennadi A. Khoudoli, Alexander Hill^¶, Colin H. Macphee^§, Ivan Pinto^§, Stephen A. Smith^§, Keith E. Suckling^§, C. Roland Wolf, and Colin N. A. Palmer^**

From the  Biomedical Research Centre,  Imperial Cancer Research Fund, Molecular Pharmacology Unit, and the ^¶ Department of Medicine, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland and ^§ GlaxoSmithKline, New Frontiers Science Park North, Third Avenue, Harlow, Essex CM19 5AW, United Kingdom

Received for publication, September 4, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The peroxisome proliferator-activated receptors (PPARs) are a family of fatty acid-activated transcription factors which control lipid homeostasis and cellular differentiation. PPAR (NR1C1) controls lipid oxidation and clearance in hepatocytes and PPAR (NR1C3) promotes preadipocyte differentiation and lipogenesis. Drugs that activate PPAR are effective in lowering plasma levels of lipids and have been used in the management of hyperlipidemia. PPAR agonists increase insulin sensitivity and are used in the management of type 2 diabetes. In contrast, there are no marketed drugs that selectively target PPAR (NR1C2) and the physiological roles of PPAR are unclear. In this report we demonstrate that the expression of PPAR is increased during the differentiation of human macrophages in vitro. In addition, a highly selective agonist of PPAR (compound F) promotes lipid accumulation in primary human macrophages and in macrophages derived from the human monocytic cell line, THP-1. Compound F increases the expression of genes involved in lipid uptake and storage such as the class A and B scavenger receptors (SRA, CD36) and adipophilin. PPAR activation also represses key genes involved in lipid metabolism and efflux, i.e. cholesterol 27-hydroxylase and apolipoprotein E. We have generated THP-1 sublines that overexpress PPAR and have confirmed that PPAR is a powerful promoter of macrophage lipid accumulation. These data suggest that PPAR may play a role in the pathology of diseases associated with lipid-filled macrophages, such as atherosclerosis, arthritis, and neurodegeneration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptors (, , and ) are ligand-activated transcription factors that control lipid and glucose homeostasis (1). They are members of the nuclear receptor family and form obligate heterodimers with the retinoid X receptor (2). PPAR¹ is highly expressed in the rodent liver, where its activation up-regulates -oxidation and thus promotes lipid clearance. PPAR agonists, such as the fibrates, are effective lipid-lowering drugs. PPAR, which is activated by 15 deoxy-^12,14PGJ₂ and the thiazolidinedione class of insulin-sensitizing drugs, is expressed particularly in adipose tissue, where it initiates the differentiation cascade (3, 4). Among its known target genes are adipocyte fatty acid-binding protein and fatty acid synthase, which are effectors of lipid accumulation during adipogenesis.

PPAR has also been studied for its role in the formation of the atherosclerotic plaque, a lesion consisting of an accumulation of lipid-laden macrophages within the intima of the arterial wall. These studies started with the observation that PPAR up-regulates CD36 (scavenger receptor-class B) expression in macrophages. This facilitates uptake of modified plasma low density lipoproteins into the macrophages potentially leading to the formation of foam cells (5, 6). However, plaque formation is also appreciated to be an inflammatory response and it has been shown that activators of PPAR inhibit the production of inflammatory cytokines such as tumor necrosis factor-, interleukin-6 and interleukin-1 (7-11). Although the PPAR dependence of these effects is controversial (12, 13), the mechanism of this inhibition appears to be via repression of AP-1, NF-B, and STAT-1 activity (8, 14, 15).

Furthermore, it has recently been shown that activation of PPAR by the thiazolidinedione, rosiglitazone, decreases scavenger receptor A expression (16) and increases ABCA1 expression (17). ABCA1 is a member of the ATP-binding cassette transporter family and it is involved in the control of the apoA1-mediated cholesterol efflux from macrophages and other peripheral lipid stores. Mutations in this protein occur in patients with Tangiers disease, a syndrome that is characterized by the pathological accumulation of cholesterol esters in many tissues. Up-regulation of ABCA1 may promote cholesterol clearance, and overall, PPAR appears to be a negative regulator of cholesterol accumulation (16, 17). Indeed, in vivo studies using animal models and preliminary clinical data have suggested that PPAR agonists oppose atheroma progression (18-22).

In contrast to PPAR and -, the function of PPAR is relatively unknown. PPAR, also known as PPAR, NUC1, and FAAR, has been shown to be expressed in a wide range of tissues, but progress in understanding the function of this protein has been hampered by the lack of selective ligands. PPAR has recently been implicated in a wide range of physiological and pathophysiological processes such as embryonic implantation, wound healing, inflammation, cancer, and osteoporosis (23-29). In this report, we show that PPAR mRNA and protein levels are dramatically elevated during macrophage differentiation and provide both genetic and pharmacological evidence that PPAR is a positive effector of lipid accumulation in human macrophage cultures. We also show that PPAR coordinates a complex pattern of gene expression controlling lipid uptake, transport, storage, metabolism, and efflux.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of Human Monocytes and in Vitro Monocyte/Macrophage Differentiation-- Human monocytes were isolated from buffy coat preparations of whole blood taken from healthy volunteers. In brief, the buffy coat was mixed with Optiprep^TM (Robbins Scientific Ltd.) in a ratio of 2.5:1 and then overlaid with a discontinuous Optiprep^TM gradient, prepared according to the reagent datasheet. Following centrifugation for 25 min at 600 × g the monocyte layer formed within the top 5-10 ml of the gradient was removed, washed with phosphate-buffered saline, and resuspended in RPMI 1640, supplemented with 2 mM glutamine and 10% human serum (Sigma). Cell viability was assessed by the ability to exclude trypan blue and was typically 95%. Monocyte purity was determined by differential counts of DiffQuik (Porvair Sciences Ltd.) stained cell preparations and was typically >95%. For monocyte-macrophage differentiation, monocytes isolated as above were resuspended in culture medium at a density of 2.5 × 10⁶/ml and seeded into 12-well tissue culture plates; medium was changed every 48 h. LDL, oxidized LDL, and lipid-depleted serum was obtained from Intracel Corporation.

Transient Transfection-- COS-1 cells were transfected by a DEAE-dextran method as previously described (30). The reporter construct pFABPLUC contains 4 copies of the peroxisome proliferator response element from the human liver FABP gene in front of the herpes simplex virus-thymidine kinase promoter, cloned immediately upstream of the cDNA encoding firefly luciferase in pGLBAS (Promega). The PPAR expression vectors contained the coding sequence for human PPAR, -, and - under the control of the enhancer/promoter of the human cytomegalovirus. PSVGal was co-transfected with each sample to act as an internal control for transfection. Cell lysates were prepared and luciferase and -galactosidase activities were assayed using kits as described by the manufacturer (Promega). Data are presented as the relative light units obtained with the luciferase assay divided by the absorbance obtained at 405 nm in the -galactosidase assays (Luc/-galactosidase).

Culture and Differentiation of THP-1 Cells-- THP-1 cells were obtained from ATCC. Cultures were grown in RPMI supplemented with 10% heat-inactivated fetal calf serum. Differentiation was initiated by the addition of 5 ng/ml PMA in the above medium. All drugs were added in Me₂SO and were replaced at intervals of 48 h. After 4 days the cells were fixed with 0.66% paraformaldehyde then stained with Oil red O (Sigma) and hematoxylin (Sigma). Quantification of lipid accumulation was achieved by extracting Oil red O from stained cells with isopropyl alcohol and measuring the optical density of the extract at 510 nm. The value obtained using a control culture was subtracted from the resulting values. The Oil red O absorbance was corrected by co-staining DNA with SYBR green dye (Molecular Probes) and quantified on a Labsystems FluoroSkan Ascent FL microplate fluorimeter. Cell number was determined from a standard curve.

Isolation of Stable Cell Lines-- The entire coding sequence of human PPAR was subcloned, in both sense and antisense orientations, into the eukaryotic expression vector pCLDN (31). The resulting plasmids were transfected into THP-1 cells using a modified DEAE-dextran procedure (32). The cells were maintained in medium containing 1 mg/ml G418 and 10% THP-1 conditioned medium, with rigorous washing procedures to remove dead cells. This was continued until cell killing stopped and robust growth was observed. We obtained six independently transfected polyclonal populations each of pCLDN (empty vector) and pCLDNPPAR (SENSE).

Western Blotting-- Cultures were lysed in SDS-polyacrylamide gel electrophoresis loading buffer and analyzed by Western blotting using standard procedures. The PPAR antiserum (obtained from Dr. David Bell) was used at a 1:2000 dilution and the anti-AFAR antiserum (obtained from Dr. John Hayes) was used at a 1:3000 dilution. A peroxidase-conjugated mouse anti-rabbit IgG antiserum (Sigma) was used as a secondary detection reagent at 1:3000 and the results were visualized using enhanced chemiluminescence (ECL+) as described by the manufacturer (Amersham Pharmacia Biotech).

Lipid Extraction and Measurement-- Cultures were treated as above and then extracted with methanol/chloroform/phosphate-buffered saline (1:1:1, v/v) containing stigmasterol as an internal standard for cholesterol. The organic phase was analyzed for cholesterol with and without saponification by GC-MS using a Finnegan ThermoQuest Trace 2000. Triglycerides were quantified colorimetrically using a kit from Sigma and triolein as a standard. Protein concentrations were determined using the Bio-Rad Protein assay reagent.

Cholesterol Efflux Assays-- THP-1 cells were differentiated with PMA in the absence or presence of 100 nM compound F for 3 days. PPAR SENSE cells were differentiated with PMA for 3 days. The cells were labeled with 2 µCi (40 nmol) of [4-¹⁴C]cholesterol (Amersham Pharmacia Biotech) in 2 ml of medium containing 10% fetal calf serum for 3 h. ApoA1 specific efflux was determined as previously described (33). For apoA1 independent efflux assays the cells were incubated a further 120 h in medium containing 10% fetal calf serum. Radioactivity release into the medium was determined by scintillation counting at 1, 4, 18, 24, 48, 72, and 120 h. All cultures incorporated similar levels (between 80 and 95%) of the labeled cholesterol. Cell viability was similar between cultures using this procedure.

RNA Extraction and Analysis-- RNA was extracted from THP-1 cells using TRIZOL reagent as recommended by the manufacturer. cDNA was synthesized from such RNA using "You-Prime Ready-To-Go" beads from Amersham Pharmacia Biotech. TAQMAN^TM real time PCR analysis was applied using prepared reagents from PerkinElmer Life Sciences. The primers and probes will be described elsewhere.² Relative levels of mRNA were calculated using the values obtained with each target gene compared with the values obtained with the 18 S ribosomal RNA probes.

Statistical Analysis-- All graphs and statistics were prepared using Graphpad Prism for the Macintosh v3.0 (Graphpad Inc., San Diego, CA). Nonlinear regression was applied using a sigmoidal response model for the dose response to compound F in transient transfection experiments (Fig. 3). p values displayed in Figs. 7, B and C, and 8E were calculated using a standard Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PPAR Accumulation in Macrophages Is Accompanied by Increases in the Expression of PPAR-- To examine the role of PPARs in macrophage lipid accumulation we measured the levels of PPAR protein and mRNA during the differentiation of primary human macrophages in vitro. We confirmed previous observations that PPAR protein levels are increased during this process (5, 6, 8, 10) (Fig. 1A). Interestingly, we also found that PPAR levels are markedly increased during macrophage differentiation. PPAR protein was also detectable, but was not greatly altered during differentiation. This pattern of regulation was confirmed at the mRNA level (Fig. 1B).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   PPAR expression is increased during macrophage differentiation. A, protein was prepared from cultures of human monocytes at day 0, 1, and 4 after the plating. The lysates were analyzed by Western blotting for the presence of PPAR, -, and -. B, RNA was prepared from the above cultures. These RNAs were analyzed for PPAR, -, and - expression using TAQMANTM procedures. All results show the mean ± S.E., n = 3.

PPAR Activation Negatively Regulates Lipid Accumulation in Macrophages-- We then examined the effect of rosiglitazone, a highly selective PPAR agonist, on the accumulation of lipid in primary human macrophages under normal culture conditions (10% human serum) (Fig. 2, A versus B). We observed that rosiglitazone (1 µM) partially inhibited the accumulation of lipid under such conditions. We also found that rosiglitazone did not stimulate lipid accumulation in primary macrophages in lipid-depleted serum alone, or supplemented with 100 µg/ml normal or oxidized low density lipoproteins (Fig. 2C, nLDL and oxLDL, respectively).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Activation of PPAR does not promote lipid accumulation in human macrophages. A, macrophages cultured in 10% human serum and stained with Oil red O. B, rosiglitazone (1 µM) inhibits lipid accumulation in macrophages cultured with 10% human serum. C, rosiglitazone (1 µM) inhibits lipid accumulation in the presence of oxidized LDL (100 µg/ml). All results show the mean ± S.E., n = 3.

Activation of PPAR Promotes Lipid Accumulation in Primary Human Macrophages-- In view of the marked induction of PPAR during human macrophage differentiation, we investigated whether it may be a regulatory factor in the process of lipid accumulation. Investigations into the role of PPAR in lipid metabolism have been hampered by the lack of PPAR selective agonists. A series of compounds has recently been described as having selectivity for PPAR (33, 34), and we have synthesized one of these compounds (compound F) that has an EC₅₀ of 2 nM for hPPAR, 400 nM for hPPAR, and >1 µM for hPPAR (Fig. 3A). Interestingly, this compound has no selectivity between mouse PPAR and - (data not shown). Human macrophages were therefore treated with compound F (100 nM) in the presence of serum, resulting in a profound increase in lipid accumulation (Fig. 3B). The formation of foam cells is thought to be promoted by modified lipoproteins in vivo. We therefore tested the ability of compound F to facilitate lipid accumulation in the presence of oxidized LDL (Fig. 3C). Oxidized LDL is a stimulus for lipid accumulation in human macrophages and addition of increasing concentrations of compound F stimulates further increases in lipid accumulation. The dose response of this lipid accumulation was very similar to the concentrations required for activation of PPAR in COS-1 cells and occurred at concentrations well below those required for the activation of PPAR and -. Importantly, lipid loading was not observed with normal LDL or lipid-depleted serum at any concentration of compound F (data not shown). This demonstrates that the action of compound F is dependent on the nature and availability of the lipid donors in the culture medium.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   A selective agonist of human PPAR promotes lipid accumulation in human monocytes. A, dose response of activation of human PPARs by compound F in COS-1 cells. COS-1 cells were transfected with a PPAR expression vector and a reporter construct containing the luciferase cDNA under the control of the PPRE found in the human FABP gene. B, compound F (100 nM) stimulates macrophage lipid accumulation in the presence of serum. C, low concentrations of compound F stimulate lipid accumulation in the presence of oxidized LDL supplemented (100 µg/ml) and lipid-depleted serum. All results show the mean ± S.E., n = 3.

PPAR Promotes Lipid Accumulation in the Monocytic Cell Line THP-1-- To obtain further evidence that PPAR is involved in macrophage lipid accumulation we utilized a cell line model where the levels of PPAR could be modulated genetically. The human monocytic cell line, THP-1, can be differentiated into adherent macrophage-like cells by the addition of phorbol ester to the culture medium. Measurement of PPAR mRNA levels following phorbol ester treatment showed that PPAR expression remained unchanged during differentiation (Fig. 4A). The levels of both PPAR and - mRNA were greatly increased upon differentiation (50- and 34-fold, respectively). These findings are similar to those obtained during the differentiation of primary macrophages (Fig. 1). To determine whether the PPARs were involved in lipid accumulation, THP-1 cells were treated with activators of PPAR, -, and - in the presence of serum. No change in lipid accumulation was observed on the addition of the PPAR activator, Wy14,643, or the PPAR activator, rosiglitazone (Fig. 4B). However, when THP-1 cells were treated with compound F, a marked increase in intracellular lipid was observed (Fig. 4B). The EC₅₀ of compound F for this effect was higher than that observed for the primary monocytes (~400 nM, Fig. 4C). The effect of compound F was further enhanced by the addition of the RXR agonist, LG100268 (Fig. 4D); further supporting the action of a PPAR/RXR heterodimer in this process. There was a high degree of similarity in the findings with those obtained in primary macrophages indicating that THP-1 cells provide a good model for studying the role of PPAR in macrophage lipid accumulation.

View larger version (60K): [in this window] [in a new window]   Fig. 4.   Activation of PPAR promotes lipid accumulation in a human monocytic cell line. A, THP-1 cells were treated with PMA for 3 days and analyzed for PPAR mRNA levels by TAQMANTM procedures. B, the effects of PPAR agonists on lipid accumulation were assessed by Oil red O staining. Me2SO (0.5%), rosiglitazone (40 µM), Wy14,463 (25 µM), and compound F (1 µM). C, low concentrations of compound F promote lipid accumulation. The effects of increasing concentrations of compound F on lipid accumulation are shown. D, compound F and LG100268 co-operate in lipid accumulation. Shown is the lipid accumulation after treatment with solvent alone (Me2SO), LG100268 (LG268,10 nM), compound F (CompF, 1 µM), or a combination of LG100268 and compound F (LG+F). DMSO, dimethyl sulfoxide.

PPAR Is a Potent Regulator of Genes Involved in Lipid Accumulation-- The lipid accumulation phenotype observed with compound F may reflect regulation of a number of aspects of lipid transport and metabolism in these cells. Recent studies have shown that rosiglitazone modulates lipid uptake by transcriptional regulation of the class B scavenger receptor CD36, but that this is balanced by increases in efflux via ABCA1 and decreases in the class A scavenger receptor, SRA (16). We therefore investigated the effects of compound F on the expression of a range of genes known to determine lipid balance in macrophages. We performed a dose-response experiment with PPAR selective concentrations of compound F and compared these treatments to a saturating dose of rosiglitazone (500 nM) (Fig. 5A). We observed that CD36 is effectively induced (6-fold) by 10 nM compound F, and that this induction is greater than that seen with 500 nM rosiglitazone (2-3-fold). This suggests that CD36 is a target gene for both PPAR and PPAR. The class A scavenger receptor was also slightly increased by 10 nM compound F (2.5-fold), whereas rosiglitazone does not modify the expression of this gene. The gene encoding the adipose-type fatty acid-binding protein (AFABP) is highly regulated by both rosiglitazone and 10 nM compound F, again showing an overlapping set of target genes. However, the gene encoding the lipid-vesicle coat protein, adipophilin is very selectively regulated by compound F (10-fold versus 3-fold with rosiglitazone). The selectivity for activation of SRA, CD36, and adipophilin was confirmed in a time course study (Fig. 5B) where it was clear that compound F mediates a larger and more sustained induction of these gene products relative to rosiglitazone. The induction of SRA was very modest but sustained, in contrast no significant induction with rosiglitazone was seen at any time point. CD36 induction by compound F appeared to be diminished at longer time points but adipophilin was highly induced throughout the experiment.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   PPAR regulates genes involved in lipid uptake, transport, storage, and efflux. A, PMA-differentiated THP-1 cells were treated with increasing concentrations of compound F (CF) or 500 nM rosiglitazone (Ros) for 48 h and analyzed for several mRNA levels by TAQMANTM procedures. B, PMA differentiated THP-1 cells were treated with 100 nM compound F or 500 nM rosiglitazone and RNA was prepared at 2, 4, 8, 16, 48, 72, and 125 h and analyzed for several mRNA levels by TAQMANTM procedures. The values obtained relative to Me2SO (DMSO) at each time point are shown. All results show the mean ± S.E., n = 3.

Overexpression of PPAR Results in Increased Lipid Accumulation-- To obtain evidence for a direct role for PPAR in macrophage lipid accumulation, we generated THP-1 sublines that overexpressed PPAR (PPARSENSE). Constitutive expression of PPAR in the PPARSENSE cells was confirmed by Western blotting (Fig. 6A). In agreement with the effects of the PPAR agonist, PPAR SENSE cell lines were extremely lipid-laden when compared with the wild type cells, even in the absence of added PPAR ligand (but in the presence of serum) (Fig. 6B). This response is very rapid with very obviously lipid-laden cells appearing 3 days after differentiation, whereas compound F promotes lipid accumulation much more slowly, only becoming visible after 7 to 10 days of treatment (data not shown). We used gas chromatography/mass spectrophotometry to measure the levels of free and esterified cholesterol present in THP-1 cells differentiated with PMA with and without compound F (100 nM), and in differentiated PPARSENSE cells (Fig. 6C). We found that compound F treatment resulted in a 50% increase in cholesterol esters compared with the untreated cells, however, the levels of free cholesterol were seen to be increased in the PPARSENSE cells. This difference may lie in the kinetics of lipid accumulation in the SENSE cells where visible lipid accumulation occurs much more rapidly than in the compound F-treated THP-1 cells. This may deplete the cells of cholesterol acceptors within the cell thus resulting in an accumulation of free cholesterol. This hypothesis is supported by the observation that triglyceride levels are massively increased in the PPARSENSE cells (Fig. 6D). Interestingly, triglyceride accumulation in conjunction with cholesterol is a characteristic of human rather than rodent macrophages (35).

View larger version (52K): [in this window] [in a new window]   Fig. 6.   Overexpression of PPAR promotes lipid accumulation. A, Western blotting reveals high levels of PPAR protein in PPARSENSE cell lines. Constant protein loading is confirmed by an antibody to aflatoxin reductase (AFAR) B, THP-1 cells and PPAR SENSE cells were treated with PMA for 3 days, fixed, and stained with Oil red O. C, THP-1 cells were treated with PMA and Me2SO or 100 nM compound F for 10 days. PPAR cells were treated with PMA for 10 days. Lipids were extracted and analyzed for cholesterol by GC-MS; D, lipids were analyzed for triglyceride content. E, cells growing in medium without PMA were analyzed for expression of the AFABP by TAQMANTM procedures. E, cells growing in medium without PMA were analyzed for expression of adipophilin by TAQMANTM procedures. All results show the mean ± S.E., n = 3. DMSO, dimethyl sulfoxide.

Overexpression of PPAR Modulates Expression of Genes Involved in Lipid Transport and Storage-- The nonstimulated PPARSENSE cells contain high constitutive levels of AFABP and adipophilin, with AFABP present at levels over 100 times those observed in the untransfected cells (Fig. 6, E and F). This suggests that the nonstimulated PPARSENSE cells have a certain degree of signaling through the overexpressed PPAR protein and provides further confirmation of the role for PPAR in the regulation of these genes. These two genes are the most sensitive to compound F (Fig. 5) and it was notable that none of the other target genes (including CD36) were elevated in nonstimulated PPARSENSE cells (data not shown). This reinforces the concept of a hierarchy of gene targets in PPAR biology. Adipophilin is a marker of lipid loading in macrophages (36) and forms a major component of the coating for the intracellular lipid vesicles. Large amounts of this protein would therefore be required to form the vesicles seen in Fig. 6B (37). In contrast to adipocytes, perilipin is barely expressed in THP-1 cells (data not shown). Perilipin is the final coating for lipid storage in adipocytes and such perilipin-bound vesicles are susceptible to mobilization by PKA. Therefore, it is possible that PPAR may induce a lipid storage pool that is less susceptible to lipolytic signals.

PPAR Represses a Subset of Genes Involved in Cholesterol Efflux-- Upon activation of THP-1 cells we found that cholesterol 27-hydroxylase (CYP27) and apolipoprotein E (apoE) were repressed by compound F (Fig. 7, A and C). This repression was also seen in the activated and lipid-filled PPARSENSE cells in the absence of compound F (Fig. 7, B and D). ApoE and CYP27 are important mediators of lipid efflux from macrophages (38), and reduced expression may contribute to the defect in lipid clearance observed in the PPAR overexpressing cells. However, during the preparation of this manuscript there has been a report of the up-regulation of macrophage ABCA1 by the PPAR agonist, GW501516, and a subsequent increase in apoA1-mediated efflux from cells treated with this compound (33). Our results confirm that the expression of ABCA1 is increased by agonists of PPAR (Fig. 5A), however, the finding that apoE and CYP27 gene expression is greatly diminished suggests that total cholesterol efflux may not be increased by these compounds. We therefore compared the effects of compound F and PPAR overexpression on the apoA1-dependent cholesterol efflux and total efflux of radioactive cholesterol into medium containing normal serum. In these studies we found that apoA1-specific efflux was increased by compound F treatment and PPAR overexpression (Fig. 7E) in agreement with our observation that ABCA1 is up-regulated and with the study of Oliver et al. (33). In contrast, total efflux from the cells was significantly attenuated by compound F treatment or PPAR overexpression (Fig. 7F). This data supports our gene expression data in suggesting that PPAR may have opposing effects on different lipid efflux pathways. The rank order of efflux-inhibition correlated well with the rate of lipid accumulation i.e. SENSE > compound F > control THP-1.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Cholesterol efflux is modulated selectively by PPAR. PMA-differentiated THP-1 cells were treated with increasing concentrations of compound F (CF) or 500 nM rosiglitazone (Ros) for 48 h and analyzed for cholesterol 27-hydroxylase (A) and apolipoprotein E (C) mRNA levels by TAQMANTM procedures. PMA-differentiated THP-1 and PPARSENSE cells were analyzed for cholesterol 27-hydroxylase (B) and apolipoprotein E (D) mRNA levels by TAQMANTM procedures. E, THP-1 cells were differentiated with PMA in the absence or presence of 100 nM compound F for 3 days. PPARSENSE cells were differentiated with PMA for 3 days. The cells were labeled with 14C cholesterol and apoA1-specific efflux was determined. Shown is mean ± S.E., n = 3. F, cells were labeled with [14C]cholesterol as above. Efflux was measured in the presence of serum between 18 and 120 h. No significant differences were observed between 1 and 18 h (data not shown). Results shown are the mean ± S.E., n = 6. DMSO, dimethyl sulfoxide.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have demonstrated that the expression of PPAR is increased dramatically during the differentiation of human primary macrophages and upon differentiation of THP-1 cells with phorbol ester. This has suggested a potential role for PPAR in the modulation of atherosclerosis and inflammation. The awareness of coincident expression of PPAR and PPAR is important in the interpretation of studies exploring macrophage biology that have used poorly selective PPAR agonists (5, 6, 10, 39). In the current study we have used highly selective agonists to demonstrate that both PPAR and PPAR differentially regulate the lipid accumulation in macrophages, with PPAR activation promoting lipid accumulation and PPAR promoting lipid clearance. These findings are in contrast with studies that suggested PPAR may promote macrophage lipid accumulation; however, these studies utilized compounds that are not as selective for PPAR as rosiglitazone (5, 6). Indeed, several publications, which were published during the preparation of this manuscript (7, 16, 17), support our finding that PPAR activation by rosiglitazone opposes lipid accumulation in macrophages. This role for PPAR in lipid clearance is in distinct contrast to its role in adipocyte differentiation, but is similar to the proposed role for PPAR in the clearance of lipid from pancreatic  cells (40). We have found that PPAR selectively promotes the accumulation of lipid in both THP-1 cells and ex vivo human macrophage cultures. This lipid accumulation occurs because of a complex regulation of gene products that control many aspects of lipid homeostasis (Fig. 8). PPAR controls lipid uptake, via the class A and class B scavenger receptors (SR-A, CD36). Transport is mediated by increases in lipid-binding proteins such as AFABP. Storage is facilitated by the production of large amounts of lipid vesicle coating proteins such as adipophilin, while metabolism of cholesterol to bile acids is regulated by the repression of CYP27. Lipid efflux is modulated in a contrasting manner by the induction of ABCA1 and the repression of apolipoprotein E. It is clear that this list of PPAR-regulated genes is incomplete and that the dramatic lipid storage phenotype that we have observed is determined by a complex interplay of such target genes. Importantly, we have characterized the novel repression of two gene products that are intimately involved in lipid storage disorders, i.e. apoE and CYP27. It is clear that both of these proteins are important in human lipid homeostasis. Indeed, genetic studies have shown that functional polymorphisms of the human apoE gene are associated with atherosclerosis (41-44) and mutations in the CYP27 gene are seen in patients with cerebrotendinous xanthomatosis (45). This is a severe cholesterol storage disorder where cholesterol is deposited throughout the body, but is primarily targeted to the vasculature and brain. The metabolism of cholesterol by this enzyme in the macrophage directs cholesterol to bile acid synthesis and export, independent of apoA1 lipid acceptors (46-49). Therefore the profound repression of CYP27 and apoE expression by activation or overexpression of PPAR might contribute to the lipid accumulation observed in these cells in culture. Importantly, although PPAR and PPAR have some common target genes, apoE and CYP27 are not repressed by PPAR agonists such as rosiglitazone. PPAR agonists have been consistently shown to reduce the atherogenic profile of serum lipids (50, 51) and have inhibitory effects on atheroma formation (18-22). We and others have shown that this is reflected in the modest ability of rosiglitazone to promote lipid clearance in human monocytes in vitro and it is likely that the anti-inflammatory nature of PPAR agonists also contribute to inhibition of atherosclerotic processes. In contrast, PPAR agonists have been shown to have complex effects on serum lipids in different animal models, with a common feature of raising total serum cholesterol (33, 50, 52). The relevance to man of studies of PPARs on lipids in small animals is often limited. One of these studies, using the highly selective PPAR agonist, GW501516, has also shown a potent raising of serum high density lipoprotein and lowering of serum triglycerides in obese rhesus monkeys (33). The mechanism of triglyceride lowering by GW501516 was distinct from the mechanism of lipid lowering seen in the use of PPAR ligands such as bezafibrate. The repression of apoC-III expression by fibrates is considered to be an important factor in lipid lowering and bezafibrate treatment of obese rhesus monkeys produces marked decreases in serum apoC-III, whereas GW501516 produced a marked increase in serum apoC-III. GW501516 has no effects on apoC-III gene expression in cultured human hepatocytes and the mechanism of lipid lowering by GW501516 is yet to be determined. It is therefore important that future studies address the effects that PPAR agonists have on both the processing of lipid in specific lipid depots, and whole body lipid balance. However, it is clear that PPAR agonists behave differently in different animal models and such studies will have to be chosen with great care. Our study has focused on the pharmacology and molecular biology of the human receptor in human cells and we have demonstrated that PPAR mediates a complex program of gene expression for the control of lipid accumulation in human macrophages. Further investigations will be required to determine the usefulness of PPAR agonists and antagonists in the management of human metabolic and vascular disease.

View larger version (48K): [in this window] [in a new window]   Fig. 8.   PPAR modulates many aspects of lipid homeostasis in macrophages. Shown is a schematic of various processes, which are controlled by PPAR, and determine the lipid storage phenotype.

	ACKNOWLEDGEMENTS

We thank Gary Moore for help with the TAQMAN reagents, Dr. David Bell for the antiserum for PPAR, and Professor John Hayes for the antiserum against aflatoxin reductase. We also thank Dr. Roger Tatoud for help with graphic design (Fig. 8) and helpful discussions during the preparation of this manuscript.

	FOOTNOTES

^* This work was supported by a grant from SmithKline Beecham Pharmaceuticals (to H. V.) and by The British Heart Foundation (to G. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 44-0-1382-632744; Fax: 44-0-1382-669993; E-mail: palmerc@icrf.icnet.uk.

Published, JBC Papers in Press, September 13, 2001, DOI 10.1074/jbc.M108482200

² L. Patel and C. Macphee, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; RXR, 9-cis-retinoic acid receptor; FABP, fatty acid-binding protein; PMA, phorbol 12-myristate 13-acetate; apo, apolipoprotein; LDL, low density lipoprotein; ABC, ATP binding cassette; SR, scavenger receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				B. Fan, S. Ikuyama, J.-Q. Gu, P. Wei, J.-i. Oyama, T. Inoguchi, and J. Nishimura Oleic acid-induced ADRP expression requires both AP-1 and PPAR response elements, and is reduced by Pycnogenol through mRNA degradation in NMuLi liver cells Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E112 - E123. [Abstract] [Full Text] [PDF]

				D. C. Berry and N. Noy All-trans-Retinoic Acid Represses Obesity and Insulin Resistance by Activating both Peroxisome Proliferation-Activated Receptor {beta}/{delta} and Retinoic Acid Receptor Mol. Cell. Biol., June 15, 2009; 29(12): 3286 - 3296. [Abstract] [Full Text] [PDF]

				B. Payre, P. de Medina, N. Boubekeur, L. Mhamdi, J. Bertrand-Michel, F. Terce, I. Fourquaux, D. Goudouneche, M. Record, M. Poirot, et al. Microsomal antiestrogen-binding site ligands induce growth control and differentiation of human breast cancer cells through the modulation of cholesterol metabolism Mol. Cancer Ther., December 1, 2008; 7(12): 3707 - 3718. [Abstract] [Full Text] [PDF]

				N. Dobrosi, B. I. Toth, G. Nagy, A. Dozsa, T. Geczy, L. Nagy, C. C. Zouboulis, R. Paus, L. Kovacs, and T. Biro Endocannabinoids enhance lipid synthesis and apoptosis of human sebocytes via cannabinoid receptor-2-mediated signaling FASEB J, October 1, 2008; 22(10): 3685 - 3695. [Abstract] [Full Text] [PDF]

				Y. Takata, J. Liu, F. Yin, A. R. Collins, C. J. Lyon, C.-H. Lee, A. R. Atkins, M. Downes, G. D. Barish, R. M. Evans, et al. PPAR{delta}-mediated antiinflammatory mechanisms inhibit angiotensin II-accelerated atherosclerosis PNAS, March 18, 2008; 105(11): 4277 - 4282. [Abstract] [Full Text] [PDF]

				T. D. Russell, C. A. Palmer, D. J. Orlicky, E. S. Bales, B. H.-J. Chang, L. Chan, and J. L. McManaman Mammary glands of adipophilin-null mice produce an amino-terminally truncated form of adipophilin that mediates milk lipid droplet formation and secretion J. Lipid Res., January 1, 2008; 49(1): 206 - 216. [Abstract] [Full Text] [PDF]

				L. Ravaux, C. Denoyelle, C. Monne, I. Limon, M. Raymondjean, and K. El Hadri Inhibition of Interleukin-1{beta}-Induced Group IIA Secretory Phospholipase A2 Expression by Peroxisome Proliferator-Activated Receptors (PPARs) in Rat Vascular Smooth Muscle Cells: Cooperation between PPAR{beta} and the Proto-Oncogene BCL-6 Mol. Cell. Biol., December 1, 2007; 27(23): 8374 - 8387. [Abstract] [Full Text] [PDF]

				T. D. Russell, C. A. Palmer, D. J. Orlicky, A. Fischer, M. C. Rudolph, M. C. Neville, and J. L. McManaman Cytoplasmic lipid droplet accumulation in developing mammary epithelial cells: roles of adipophilin and lipid metabolism J. Lipid Res., July 1, 2007; 48(7): 1463 - 1475. [Abstract] [Full Text] [PDF]

				L. Sarov-Blat, R. S. Kiss, B. Haidar, N. Kavaslar, M. Jaye, M. Bertiaux, K. Steplewski, M. R. Hurle, D. Sprecher, R. McPherson, et al. Predominance of a Proinflammatory Phenotype in Monocyte-Derived Macrophages From Subjects With Low Plasma HDL-Cholesterol Arterioscler. Thromb. Vasc. Biol., May 1, 2007; 27(5): 1115 - 1122. [Abstract] [Full Text] [PDF]

				E. Dyroy, T. H. Rost, R. J. Pettersen, B. Halvorsen, O. A. Gudbrandsen, T. Ueland, Z. Muna, F. Muller, J. E. Nordrehaug, P. Aukrust, et al. Tetradecylselenoacetic Acid, a PPAR Ligand With Antioxidant, Antiinflammatory, and Hypolipidemic Properties Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): 628 - 634. [Abstract] [Full Text] [PDF]

				J. D. Brown and J. Plutzky Peroxisome Proliferator Activated Receptors as Transcriptional Nodal Points and Therapeutic Targets Circulation, January 30, 2007; 115(4): 518 - 533. [Abstract] [Full Text] [PDF]

				L. Piqueras, A. R. Reynolds, K. M. Hodivala-Dilke, A. Alfranca, J. M. Redondo, T. Hatae, T. Tanabe, T. D. Warner, and D. Bishop-Bailey Activation of PPAR{beta}/{delta} Induces Endothelial Cell Proliferation and Angiogenesis Arterioscler. Thromb. Vasc. Biol., January 1, 2007; 27(1): 63 - 69. [Abstract] [Full Text] [PDF]

				R. Nielsen, L. Grontved, H. G. Stunnenberg, and S. Mandrup Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery Mol. Cell. Biol., August 1, 2006; 26(15): 5698 - 5714. [Abstract] [Full Text] [PDF]

				A. G. Smith and G. E. O. Muscat Orphan nuclear receptors: therapeutic opportunities in skeletal muscle Am J Physiol Cell Physiol, August 1, 2006; 291(2): C203 - C217. [Abstract] [Full Text] [PDF]

				T. Yamaguchi, S. Matsushita, K. Motojima, F. Hirose, and T. Osumi MLDP, a Novel PAT Family Protein Localized to Lipid Droplets and Enriched in the Heart, Is Regulated by Peroxisome Proliferator-activated Receptor {alpha} J. Biol. Chem., May 19, 2006; 281(20): 14232 - 14240. [Abstract] [Full Text] [PDF]

				K. T. Dalen, S. M. Ulven, B. M. Arntsen, K. Solaas, and H. I. Nebb PPAR{alpha} activators and fasting induce the expression of adipose differentiation-related protein in liver J. Lipid Res., May 1, 2006; 47(5): 931 - 943. [Abstract] [Full Text] [PDF]

				J. Lopez-Soriano, C. Chiellini, M. Maffei, P. A. Grimaldi, and J. M. Argiles Roles of Skeletal Muscle and Peroxisome Proliferator-Activated Receptors in the Development and Treatment of Obesity Endocr. Rev., May 1, 2006; 27(3): 318 - 329. [Abstract] [Full Text] [PDF]

				K. Nadra, S. I. Anghel, E. Joye, N. S. Tan, S. Basu-Modak, D. Trono, W. Wahli, and B. Desvergne Differentiation of Trophoblast Giant Cells and Their Metabolic Functions Are Dependent on Peroxisome Proliferator-Activated Receptor {beta}/{delta} Mol. Cell. Biol., April 15, 2006; 26(8): 3266 - 3281. [Abstract] [Full Text] [PDF]

				B. H.-J. Chang, L. Li, A. Paul, S. Taniguchi, V. Nannegari, W. C. Heird, and L. Chan Protection against Fatty Liver but Normal Adipogenesis in Mice Lacking Adipose Differentiation-Related Protein Mol. Cell. Biol., February 1, 2006; 26(3): 1063 - 1076. [Abstract] [Full Text] [PDF]

				U. Edvardsson, A. Ljungberg, D. Linden, L. William-Olsson, H. Peilot-Sjogren, A. Ahnmark, and J. Oscarsson PPAR{alpha} activation increases triglyceride mass and adipose differentiation-related protein in hepatocytes J. Lipid Res., February 1, 2006; 47(2): 329 - 340. [Abstract] [Full Text] [PDF]

				Y. Masuda, H. Itabe, M. Odaki, K. Hama, Y. Fujimoto, M. Mori, N. Sasabe, J. Aoki, H. Arai, and T. Takano ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells J. Lipid Res., January 1, 2006; 47(1): 87 - 98. [Abstract] [Full Text] [PDF]

				F. Blaschke, Y. Takata, E. Caglayan, R. E. Law, and W. A. Hsueh Obesity, Peroxisome Proliferator-Activated Receptor, and Atherosclerosis in Type 2 Diabetes Arterioscler. Thromb. Vasc. Biol., January 1, 2006; 26(1): 28 - 40. [Abstract] [Full Text] [PDF]

				A. Szanto and L. Nagy Retinoids Potentiate Peroxisome Proliferator-Activated Receptor {gamma} Action in Differentiation, Gene Expression, and Lipid Metabolic Processes in Developing Myeloid Cells Mol. Pharmacol., June 1, 2005; 67(6): 1935 - 1943. [Abstract] [Full Text] [PDF]

				J. M. Wallace, M. Schwarz, P. Coward, J. Houze, J. K. Sawyer, K. L. Kelley, A. Chai, and L. L. Rudel Effects of peroxisome proliferator-activated receptor {alpha}/{delta} agonists on HDL-cholesterol in vervet monkeys J. Lipid Res., May 1, 2005; 46(5): 1009 - 1016. [Abstract] [Full Text] [PDF]

				M. Vinals, I. Bermudez, G. Llaverias, M. Alegret, R. M. Sanchez, M. Vazquez-Carrera, and J. C. Laguna Aspirin increases CD36, SR-BI, and ABCA1 expression in human THP-1 macrophages Cardiovasc Res, April 1, 2005; 66(1): 141 - 149. [Abstract] [Full Text] [PDF]

				A. C. Li and C. K. Glass PPAR- and LXR-dependent pathways controlling lipid metabolism and the development of atherosclerosis J. Lipid Res., December 1, 2004; 45(12): 2161 - 2173. [Abstract] [Full Text] [PDF]

				N. Marx, H. Duez, J.-C. Fruchart, and B. Staels Peroxisome Proliferator-Activated Receptors and Atherogenesis: Regulators of Gene Expression in Vascular Cells Circ. Res., May 14, 2004; 94(9): 1168 - 1178. [Abstract] [Full Text] [PDF]

				R. L. Stephen, M. C. U. Gustafsson, M. Jarvis, R. Tatoud, B. R. Marshall, D. Knight, E. Ehrenborg, A. L. Harris, C. R. Wolf, and C. N. A. Palmer Activation of Peroxisome Proliferator-Activated Receptor {delta} Stimulates the Proliferation of Human Breast and Prostate Cancer Cell Lines Cancer Res., May 1, 2004; 64(9): 3162 - 3170. [Abstract] [Full Text] [PDF]

				G. Larigauderie, C. Furman, M. Jaye, C. Lasselin, C. Copin, J.-C. Fruchart, G. Castro, and M. Rouis Adipophilin Enhances Lipid Accumulation and Prevents Lipid Efflux From THP-1 Macrophages: Potential Role in Atherogenesis Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 504 - 510. [Abstract] [Full Text]

				S. M. Schmidt, K. Schag, M. R. Muller, T. Weinschenk, S. Appel, O. Schoor, M. M. Weck, F. Grunebach, L. Kanz, S. Stevanovic, et al. Induction of Adipophilin-Specific Cytotoxic T Lymphocytes Using a Novel HLA-A2-Binding Peptide That Mediates Tumor Cell Lysis Cancer Res., February 1, 2004; 64(3): 1164 - 1170. [Abstract] [Full Text] [PDF]

				M. Ricote, A. F. Valledor, and C. K. Glass Decoding Transcriptional Programs Regulated by PPARs and LXRs in the Macrophage: Effects on Lipid Homeostasis, Inflammation, and Atherosclerosis Arterioscler. Thromb. Vasc. Biol., February 1, 2004; 24(2): 230 - 239. [Abstract] [Full Text]

				I. Bildirici, C.-R. Roh, W. T. Schaiff, B. M. Lewkowski, D. M. Nelson, and Y. Sadovsky The Lipid Droplet-Associated Protein Adipophilin Is Expressed in Human Trophoblasts and Is Regulated by Peroxisomal Proliferator-Activated Receptor-{gamma}/Retinoid X Receptor J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6056 - 6062. [Abstract] [Full Text] [PDF]

				U. Dressel, T. L. Allen, J. B. Pippal, P. R. Rohde, P. Lau, and G. E. O. Muscat The Peroxisome Proliferator-Activated Receptor {beta}/{delta} Agonist, GW501516, Regulates the Expression of Genes Involved in Lipid Catabolism and Energy Uncoupling in Skeletal Muscle Cells Mol. Endocrinol., December 1, 2003; 17(12): 2477 - 2493. [Abstract] [Full Text] [PDF]

				B. Kutlu, A. K. Cardozo, M. I. Darville, M. Kruhoffer, N. Magnusson, T. Orntoft, and D. L. Eizirik Discovery of Gene Networks Regulating Cytokine-Induced Dysfunction and Apoptosis in Insulin-Producing INS-1 Cells Diabetes, November 1, 2003; 52(11): 2701 - 2719. [Abstract] [Full Text] [PDF]

				C.-H. Lee, A. Chawla, N. Urbiztondo, D. Liao, W. A. Boisvert, and R. M. Evans Transcriptional Repression of Atherogenic Inflammation: Modulation by PPAR{delta} Science, October 17, 2003; 302(5644): 453 - 457. [Abstract] [Full Text] [PDF]

				J.-C. Mamputu, N. Wiernsperger, and G. Renier Metformin inhibits monocyte adhesion to endothelial cells and foam cell formation The British Journal of Diabetes & Vascular Disease, July 1, 2003; 3(4): 302 - 310. [Abstract] [PDF]

				M. Gurnell, D. B. Savage, V. K. K. Chatterjee, and S. O'Rahilly The Metabolic Syndrome: Peroxisome Proliferator-Activated Receptor {gamma} and Its Therapeutic Modulation J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2412 - 2421. [Abstract] [Full Text] [PDF]

				C.-H. Lee, P. Olson, and R. M. Evans Minireview: Lipid Metabolism, Metabolic Diseases, and Peroxisome Proliferator-Activated Receptors Endocrinology, June 1, 2003; 144(6): 2201 - 2207. [Abstract] [Full Text] [PDF]

				J. Skogsberg, K. Kannisto, T. N. Cassel, A. Hamsten, P. Eriksson, and E. Ehrenborg Evidence That Peroxisome Proliferator-Activated Receptor Delta Influences Cholesterol Metabolism in Men Arterioscler. Thromb. Vasc. Biol., April 1, 2003; 23(4): 637 - 643. [Abstract] [Full Text] [PDF]

				A. Chawla, C.-H. Lee, Y. Barak, W. He, J. Rosenfeld, D. Liao, J. Han, H. Kang, and R. M. Evans PPARdelta is a very low-density lipoprotein sensor in macrophages PNAS, February 4, 2003; 100(3): 1268 - 1273. [Abstract] [Full Text] [PDF]

				H. Lim and S. K. Dey Minireview: A Novel Pathway of Prostacyclin Signaling--Hanging Out with Nuclear Receptors Endocrinology, September 1, 2002; 143(9): 3207 - 3210. [Abstract] [Full Text] [PDF]

				H. Okazaki, J.-i. Osuga, K. Tsukamoto, N. Isoo, T. Kitamine, Y. Tamura, S. Tomita, M. Sekiya, N. Yahagi, Y. Iizuka, et al. Elimination of Cholesterol Ester from Macrophage Foam Cells by Adenovirus-mediated Gene Transfer of Hormone-sensitive Lipase J. Biol. Chem., August 23, 2002; 277(35): 31893 - 31899. [Abstract] [Full Text] [PDF]

				O. Barbier, I. P. Torra, Y. Duguay, C. Blanquart, J.-C. Fruchart, C. Glineur, and B. Staels Pleiotropic Actions of Peroxisome Proliferator-Activated Receptors in Lipid Metabolism and Atherosclerosis Arterioscler. Thromb. Vasc. Biol., May 1, 2002; 22(5): 717 - 726. [Abstract] [Full Text] [PDF]

				J. Zhang, M. Fu, X. Zhu, Y. Xiao, Y. Mou, H. Zheng, M. A. Akinbami, Q. Wang, and Y. E. Chen Peroxisome Proliferator-activated Receptor delta Is Up-regulated during Vascular Lesion Formation and Promotes Post-confluent Cell Proliferation in Vascular Smooth Muscle Cells J. Biol. Chem., March 22, 2002; 277(13): 11505 - 11512. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/47/44258    most recent M108482200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vosper, H. Articles by Palmer, C. N. A. Search for Related Content PubMed PubMed Citation Articles by Vosper, H. Articles by Palmer, C. N. A. Social Bookmarking                      What's this?