This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marx, N. Articles by Plutzky, J. Search for Related Content PubMed PubMed Citation Articles by Marx, N. Articles by Plutzky, J.

(American Journal of Pathology. 1998;153:17-23.)
© 1998 American Society for Investigative Pathology

Short Communication

Macrophages in Human Atheroma Contain PPAR

Differentiation-Dependent Peroxisomal Proliferator-ActivatedReceptor (PPAR) Expression and Reduction of MMP-9 Activitythrough PPAR Activation in Mononuclear Phagocytes inVitro

From the Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mononuclear phagocytes play an important role in atherosclerosis and its sequela plaque rupture in part by their secretion of matrix metalloproteinases (MMPs), including MMP-9. Peroxisomal proliferator-activated receptor (PPAR), a transcription factor in the nuclear receptor superfamily, regulates gene expression in response to various activators, including 15-deoxy-^12,14-prostaglandin J₂ and the antidiabetic agent troglitazone. The role of PPAR in human atherosclerosis is unexplored. We report here that monocytes/macrophages in human atherosclerotic lesions (n = 12) express immunostainable PPAR. Normal artery specimens (n = 6) reveal minimal immunoreactive PPAR. Human monocytes and monocyte-derived macrophages cultured for 6 days in 5% human serum expressed PPAR mRNA and protein by reverse transcription-polymerase chain reaction and Western blotting, respectively. In addition, PPAR mRNA expression in U937 cells increased during phorbol 12-myristate 13 acetate-induced differentiation. Stimulation of PPAR with troglitazone or 15-deoxy-^12,14-prostaglandin J₂ in human monocyte-derived macrophages inhibited MMP-9 gelatinolytic activity in a concentration-dependent fashion as revealed by zymography. This inhibition correlates with decreased MMP-9 secretion as determined by Western blotting. Thus, PPAR is present in macrophages in human atherosclerotic lesions and may regulate expression and activity of MMP-9, an enzyme implicated in plaque rupture. PPAR is likely to be an important regulator of monocyte/macrophage function with relevance for human atherosclerotic disease.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

We therefore undertook an effort to identify endogenous inhibitors of MMP-9 expression. Work from other groups has established that activation of various nuclear hormone receptors can inhibit MMP expression through a variety of mechanisms.¹¹ Interest is growing regarding the role of peroxisomal proliferation activator receptors (PPARs), a subgroup of the nuclear receptor superfamily, as transcriptional mediators.^12,13 One of these, PPAR, has been implicated as a "master regulator" of lipid metabolism and adipogenesis; ectopic overexpression of PPAR in fibroblasts redirects these cells into an adipogenic program.^14,15 Like other nuclear receptors, PPAR contains a ligand binding domain and a central DNA binding domain, which interacts with PPAR response elements in the promoter of target genes.¹⁶ Specific activators identified thus far include both the naturally occurring prostaglandin D metabolite 15-deoxy-^12,14-prostaglandin J₂ (15 d-PGJ₂)^17-19 and the synthetic antidiabetic agent troglitazone.^20-22 The role of PPAR in nonadipocytes has received little attention, although expression had been previously noted in hematopoietic cell lines.²³ Recent work suggests PPAR stimulation can inhibit both cytokine-induced activation of macrophages²⁴ and in vitro expression of transfected promoter constructs of genes implicated in atherogenesis, including MMP-9.²⁵

The present study tested the hypotheses 1) that macrophages in human atheroma express PPAR, 2) that this novel nuclear receptor is regulated during differentiation of monocytes into macrophages, and 3) that PPAR activation can limit MMP-9 expression and enzymatic activity by these cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunohistochemistry

Surgical specimens of human carotid atherosclerotic lesions were obtained by protocols approved by the Human Investigation Review Committee at Brigham and Women's Hospital. Serial cryostat sections (5 mm) were cut, air dried onto microscopic slides, and fixed in acetone at -20°C for 5 minutes. Staining for PPAR was performed with a polyclonal rabbit anti-human PPAR peptide antibody¹⁹ (a generous gift from Dr. Mitchell Lazar, University of Pennsylvania School of Medicine, Philadelphia). Macrophages were identified by staining with anti-CD68 antibody (DAKO, Carpinteria, CA). Sections were preincubated with PBS containing 0.3% hydrogen peroxidase activity and stained for 1 hour with primary antibody diluted in PBS supplemented with 5% appropriate serum. Negative control was performed by preabsorbing the anti-PPAR antibodies with the peptide from which the antibody was derived and subsequently using these "peptide-blocked PPAR antibodies" at concentrations similar to those of experimental conditions. Finally, sections were incubated with the respective biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) followed by avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Laboratories). Antibody binding was visualized with 3-amino-9-ethyl carbazole (Vector Laboratories) or with True Blue Peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Sections were counterstained with Gill's Hematoxylin or Contrast Red (Kirkegaard & Perry Laboratories). Computer-assisted image analysis was used to quantify staining on sections using Optimas 5.2 software. Percentage area of positive staining for PPAR or CD68 in the shoulders of the plaques, defined as the intimal regions flanking the lipid core, was compared with the percentage area of positive staining in other zones of the sections.

Cell Culture

Human monocytes were isolated from peripheral blood of healthy volunteers by sequential gradient centrifugation with Lymphocyte Separation Medium (Organon Technika, Durham, NC) and One Step Monocytes (Accurate Chemical and Scientific Co., Westbury, NY). Monocytes were plated at a concentration of 3 x 10⁹ cells/L in serum-free M199 medium (BioWhittaker, Walkersville, MD) and isolated by adherence to plastic dishes at 37°C. Nonadherent cells were washed three times with Hanks' buffer (Life Technologies, Gaithersburg, MD), and the remaining adherent cells were cultured in M199 medium with 5% human serum at 37°C/5%CO₂. Medium was changed every 2 days for 6 days, and resulting cells were used as "monocyte-derived macrophages." In some experiments, monocytes were cultured for 6 days with 5% human serum in the absence or presence of PPAR activators troglitazone (provided by Parke Davis (Morris Plains, NJ) and dissolved according to manufacturer's instructions) or 15 d-PGJ₂ (CalbioChem, La Jolla, CA) at concentrations indicated. After 6 days, cells were changed to serum-free conditions, and supernatants were collected after 24 hours for further analysis. The human monocyte-like cell line U937, obtained from American Type Culture Collection (Manassas, VA), was cultured in RPMI 1640 medium (BioWhittaker) with 1% glutamine (Sigma Chemical Co., St. Louis, MO), 1% penicillin-streptomycin (Sigma) and 10% fetal calf serum.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction

Total RNA from 10⁷ cells was isolated by the single-step guanidium thiocyanate-phenol-chloroform method using RNAzol from Tel-Test (Friendswood, TX). Two micrograms of total RNA was reverse-transcribed into cDNA with 1 U/ml reverse transcriptase (Superscript, Life Technologies) at 37°C for 1 hour in standard buffer. For the amplification of PPAR cDNA, two oligonucleotide primers were designed from nucleotides +235 to 708 (a 473-bp fragment): sense primer, 5'-TCTCTCCGTAATGGAAGACC-3'; anti-sense primer, 5'-CCCCTACAGAGTATTACG-3'. The polymerase chain reaction reaction was carried out in a standard buffer (Life Technologies) with 200 ng of each primer (IDT, Coralville, CA), 33 mmol/L MgCl₂, and 0.5 U Taq polymerase (Life Technologies) for 30 cycles. Polymerase chain reaction products (10 µl/25µl reaction) were analyzed on a 2% agarose gel.

Northern Blot Analysis

Five micrograms of total RNA from undifferentiated and phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells was subjected to electrophoresis on a 1.2% agarose gel and transferred using traditional Northern blotting techniques. The membranes were ultraviolet-crosslinked, prehybridized at 42°C (50% formamide, 5x Denhardt's solution, 5x standard saline citrate, 0.5% sodium dodecyl sulfate, and 20 mmol/L salmon sperm DNA), and hybridized in the same buffer with a random primed radiolabeled ([-P³²]dCTP) Sal-1 fragment of pCMX-PPAR (generously provided by Dr. Bruce Spiegelman, Dana-Farber Cancer Institute, Boston, MA).¹⁴ The membranes were washed at 60°C in 1% sodium dodecyl sulfate/2x standard saline citrate and exposed (1–3 days, -70°C) to Kodak X-OMAT film with an intensifying screen.

Preparation of Nuclear and Cytosolic Extracts and Western Blot Analysis

For Western blot analysis, a positive control was generated by transiently transfecting a PPAR expression construct, pCMX-PPAR,¹⁴ into human skin fibroblasts using lipofectamine (Life Technologies) according to the manufacturer's protocol. Nuclear and cytosolic extracts of 10⁷ cells were prepared separately. Cells were lysed in 10 mmol/L Hepes (pH 7.9), 1.5 mmol/L MgCl₂, 10 mmol/L KCl, and 0.5% Nonidet P-40. Nuclei were pelleted at 13,000 x g for 5 minutes, and the resulting supernatant was used as the cytosolic fraction. Nuclei were lysed in 20 mmol/L Hepes (pH 7.9), 1.5 mmol/L MgCl₂, 420 mmol/L NaCl, and 0.2 mmol/L ethylenediaminetetraacetate. After centrifugation at 13,000 x g for 5 minutes, the supernatant was diluted in an equal volume of 20 mmol/L Hepes (pH 7.9), 100 mmol/L KCl, 0.2 mmol/L ethylenediaminetetraacetate, and 20% glycerol and used as nuclear extract. Protein concentration of nuclear and cystolic extracts was determined using a protein assay (Pierce, Rockford, IL). Processed samples were applied to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and protein was transferred to nitrocellulose membranes (Millipore, Bedford, MA) using semidry blotting for 1 hour, as described previously.¹⁰

Membranes were blocked overnight in Tris-buffered saline-Tween with 5% dry milk and incubated with goat anti-human PPAR monoclonal antibodies (N-20, Santa Cruz Biotechnology, San Diego, CA) for 1 hour. After washing, membranes were stained with horseradish-conjugated rabbit anti-goat monoclonal antibodies. Antigen detection was performed with a chemiluminescent detection system (NEN, Boston, MA). Similar methods were used to perform Western blots on MMP-9 in monocyte-derived macrophage supernatants using a specific rabbit anti-human MMP-9 antibody (Oncogene Science, Cambridge, MA).

Substrate Gel Zymography

Gelatinolytic activity of MMP-9 from conditioned medium (10 µl/500 µl total supernatant loaded) of monocytes or monocyte-derived macrophages was analyzed by zymography on gelatin-containing polyacrylamide.¹⁰ Equal amounts were loaded in each lane. After washing in 2.5% Triton X-100, gels were incubated overnight at 37°C in 50 mmol/L Tris-HCl (pH 7.4) containing CaCl₂ and 0.05% Brij 35. Gels were stained in 0.1% Colloidal Brilliant Blue (Sigma), 10% acetic acid, and 40% methanol for 2 hours and destained in 10% acetic acid and 40% methanol. Proteins having gelatinolytic activity were visualized as clear zones in an otherwise blue gel. Photographs of the gels were scanned by an imaging densitometer and quantified using the NIH Image 1.6 software program. To ensure that differences in protein amounts did not account for the differences seen, the zymographic data were normalized to the total amount of protein applied to each lane. Of note, the total amount of protein did not vary significantly from sample to sample.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Macrophages in Human Atheroma Contain PPAR

Analysis of human carotid atheroma (n = 12) demonstrated immunoreactive PPAR co-localizing with macrophages (Figure 1, A and C) , so identified by morphology and by staining of parallel sections with the macrophage-specific antibody anti-CD68 (data not shown). PPAR staining was mainly localized in the macrophage-rich shoulder region of the plaque (Figure 1A) , with 35 ± 5% of this area positive for PPAR and 54 ± 6% positive for macrophages, as determined by color image analysis. Quantification of staining in nonshoulder regions revealed a 2 ± 1% area positive for PPAR and 8 ± 1% for macrophages. Preabsorption of anti-PPAR antibodies with the peptide antigen abrogated staining in adjacent sections (Figure 1B) , indicating the specificity of the immunostaining. Higher-power views of PPAR-stained plaque (indicated by the rectangle in Figure 1A ) showed staining predominantly in nuclei of macrophages (Figure 1C) . Only occasional endothelial cells or smooth muscle cells in lesions showed immunoreactive PPAR (data not shown). Study of nonatheromatous arterial specimens (n = 6) showed scant PPAR in nuclei of vascular smooth muscle cells (Figure 1D) .

View larger version (106K): [in this window] [in a new window]   Figure 1. Expression of PPAR in human atherosclerotic lesions. A: Low-power view of frozen section of human carotid lesions shows immunoreactive PPAR next to the lipid core in the shoulder region with abundant macrophages present. Similar results were seen in other atherosclerotic specimens (n = 12). B: No immunoreactive PPAR is detectable in parallel sections stained with PPAR antibodies preabsorbed with the immunizing peptide, indicating that staining for PPAR in (A) and (C) is specific. Similar results were seen with immunoglobulin G controls (not shown). C: High-power view of the area indicated by the rectangle in (A) shows PPAR staining restricted to macrophage nuclei (see quantification in Results). D: In nonatherosclerotic arteries (n = 6), little PPAR could be detected, with scant staining in occasional vascular smooth muscle cells.

Cells of the Monocyte/Macrophage Lineage Express PPAR mRNA and Protein

Freshly prepared monocytes, monocyte-derived macrophages, and PMA-treated U937 cells all contained PPAR mRNA as detected by a 473-bp reverse transcription-polymerase chain reaction product (Figure 2A , upper panel). For the detection of PPAR protein, we prepared separate nuclear and cytosolic fractions of the above-mentioned cells, as well as untransfected and PPAR-transfected fibroblasts, and performed Western blot analysis. Nuclear extracts of monocytes, monocyte-derived macrophages, and differentiated U937 cells (Figure 2A , lower panel), but not cytosolic fractions (data not shown), contained PPAR protein. The identity of this band as PPAR was supported by its lack of cytosolic expression, its expected apparent molecular weight (55 kd), and co-migration with a signal in fibroblasts transfected with a PPAR expression construct. Untransfected fibroblasts demonstrate no cross-reacting band of the appropriate size (Figure 2A , lower panel).

View larger version (56K): [in this window] [in a new window]   Figure 2. A: PPAR mRNA and protein are expressed in cells of the monocyte/macrophage lineage. Upper panel: reverse transcription-polymerase chain reaction of PPAR mRNA in freshly prepared monocytes (Mo), monocyte-derived macrophages (MØ), and PMA-differentiated U937 cells (U937) reveals a cDNA fragment of the expected size. Also shown are a 100-bp DNA ladder (MW) and negative control without cDNA (Co). Lower panel: Western blot analysis of PPAR protein expression in nuclear extracts of freshly prepared monocytes (Mo), monocyte-derived macrophages (MØ), and U937 cells (U937) reveals a band of the appropriate size. The identity of this band is confirmed by co-migration with a band seen in PPAR-transfected human skin fibroblasts (+) but not in similar but untransfected fibroblasts (-). All results shown were reproduced in three independent experiments. B (right): PPAR mRNA expression in undifferentiated (Undiff.) and PMA-differentiated U937 cells (Diff.), as shown by Northern blot analysis. Differentiated U937 cells show increased PPAR mRNA expression compared with undifferentiated cells. B (left): Ethidium bromide staining demonstrates equal loading of intact RNA. Results shown were reproduced in three independent experiments.

Increased PPAR Expression during PMA-Induced Differentiation of U937 Cells

To investigate further the regulation of PPAR during differentiation of cells of the monocytic lineage, U937 cells were stimulated with PMA (10 µg/L) for 12 hours, and Northern blotting was performed. This treatment increased PPAR mRNA expression (Figure 2B) . Western blot analysis showed a parallel rise in protein levels in nuclear fractions of PMA-treated U937 cells (data not shown). Neither undifferentiated nor differentiated U937 cells demonstrate PPAR in the cytosol (data not shown).

PPAR Activators Troglitazone and PGJ₂ Decrease Both Protein Levels and Gelatinolytic Activity of MMP-9 Secreted from Monocyte-Derived Macrophages

To elucidate the functional relevance of PPAR activation, we investigated the effect of the selective PPAR activators troglitazone and 15 d-PGJ₂ on MMP-9 activity in monocyte-derived macrophages. Monocytes were cultured initially in human serum and then transferred to serum-free medium in the absence or presence of troglitazone or 15 d-PGJ₂. Secreted MMP-9 gelatinolytic activity was then measured using substrate zymography. As previously reported, culture of monocytes for 6 days with human serum substantially increases gelatinolytic activity in the supernatant compared with freshly prepared monocytes (Figure 3A) . Concurrent treatment of monocytes with troglitazone or 15 d-PGJ₂ in serum-free medium during this transition toward monocyte-derived macrophages inhibited the increase in gelatinolytic activity in a concentration-dependent manner (Figure 3A) . Quantification of gelatinolytic areas by densitometry revealed a reduction of MMP-9 activity after treatment with troglitazone (1 µM and 5 µM) to 73.5 ± 1.9% and 53.3 ± 12.2% (P < 0.01), respectively, and with 15 d-PGJ₂ (1 µM and 5 µM) to 79.0 ± 14.6% and 45.5 ± 8.9% (P < 0.01), respectively, in both cases relative to control cells (n = 3). Similar results were seen in U937 cells (data not shown). Treatment with either troglitazone or 15 d-PGJ₂ after 6 days of culture in human serum had no effect (data not shown), suggesting that PPAR activation was required during differentiation for inhibition of MMP-9 levels/activity to occur. The identity of the observed gelatinolytic area was confirmed by the mobility of the band and co-migration with the known 92-kd gelatinolytic activity in PMA-treated human fibroblast supernatants (Figure 3A) . Treatment of monocyte-derived macrophages with either PPAR activator, troglitazone or 15 d-PGJ₂, reduces supernatant MMP-9 protein levels from monocyte-derived macrophages (Figure 3B) . Co-migration with the known MMP-9 band of supernatants from PMA-stimulated vascular smooth muscle cells confirms identity of the detected band (Figure 3B) .

View larger version (42K): [in this window] [in a new window]   Figure 3. A: PPAR activators decrease MMP-9 gelatinolytic activity in supernatants from monocyte-derived macrophages (MØ) in a concentration-dependent fashion, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Monocytes were cultured in 5% human serum in the presence or absence of troglitazone or 15 d-PGJ2 as indicated. Supernatants of PMA-treated fibroblasts served as a control (Co). Gelatinolytic activity of freshly prepared monocytes is also shown (Mo). Equal amounts of lysates were loaded. Results shown were reproduced in three independent experiments. B: PPAR activators decrease MMP-9 protein levels in supernatants from monocyte-derived macrophages (MØ). Vascular smooth muscle cells treated with PMA at 50 µg/L served as a control (Co). Supernatants from freshly prepared monocyte supernatant (Mo) are also shown. Similar results were reproduced in three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Coronary atherosclerosis is typically a diffuse process.²⁶ Nevertheless, despite systemic risk factors, such as low-density lipoprotein levels, and the presence of macrophages and inflammatory cytokines in most plaques, some arterial plaques rupture, whereas others do not. The factors accounting for this variability are unclear. Certainly, monocytes/macrophages are intimately involved in plaque rupture.^2,27 As monocytes differentiate in the subintima into macrophages and foam cells, the atherogenic microenvironment influences transcriptional regulation of genes, the products of which will determine the natural history of the lesion.¹ MMPs furnish one example of proteins, the induction and expression of which by monocytes/macrophages likely contributes to subsequent plaque rupture.² Several lines of evidence indicate that lesional macrophages synthesize MMPs de novo.^28,29. These proteins are enzymatically active, as shown by zymographic analysis of human arterial specimens.³⁰ Secretion of these MMPs, with MMP-9 prominent among them, favors destabilization of the plaque's fibrous cap.¹

Our findings suggest that PPAR, as a nuclear transcription factor present in lesional macrophages, may inhibit MMP expression and activity. Furthermore, PPAR may control expression of other target genes within the arterial wall, thus modulating a cascade of responses in monocytes/macrophages after activation by its endogenous or synthetic ligand(s).

15 d-PGJ₂, a naturally occurring ligand for PPAR, likely interacts with monocytes/macrophages. J₂ prostanoids, and the immediate upstream precursors of 15 d-PGJ₂, are found in vivo.³¹ Prostaglandins themselves are synthesized from fatty acids, with arachadonic acid as the primary source. 15 d-PGD₂, the major prostaglandin in most tissues, is converted to 15 d-PGJ₂.³² This process requires 15 d-PGD₂ synthetase, an enzyme produced primarily by macrophages and other antigen-presenting cells.³³ Thus, 15 d-PGJ₂, acting through PPAR, may be an important regulator of macrophage function. Troglitazone, a synthetic PPAR activator, is currently in clinical use as an antidiabetic agent.^34,36 The implications of its effects on inhibiting macrophage MMP-9 matrix degradation merit further consideration in a clinical context.

A very recent report showed that the PPAR activator 15 d-PGJ₂ inhibits expression of an MMP-9 promoter luciferase construct when transfected into U937 cells.²⁵ Our findings support a critical role for PPAR in atherosclerosis, demonstrating its presence in monocytes/macrophages of human atheroma and increased expression during differentiation in vitro. Furthermore, the present study of MMP-9 gelatinolytic activity illustrates directly the functional relevance of PPAR in these cells. These results establish a rationale for further study of PPAR in monocyte/macrophage biology, particularly in the context of atherosclerosis.

	Acknowledgements

 
We thank Eugenia Schvartz for her expert technical assistance and our colleagues in the Vascular Medicine and Atherosclerosis Unit for their insightful technical and intellectual advice.

	Footnotes

 
Address reprint requests to Jorge Plutzky, Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115. E-mail: jplutzky{at}bics.bwh.harvard.edu

Supported by Deutsche Forschungsgemeinschaft Grant MA 2047/1-1 (to NM) and National Institutes of Health Grants HL 03107 and P50HL56985 (to JP), and P50HL56985 and R37HL34636 (to PL).

Accepted for publication April 2, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Libby P, Geng Y, Aikawa M, Schoenbeck U, Mach F, Clinton S, Sukhova G, Lee R: Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol 1996, 7:330-335[Medline]
2. Dollery C, McEwan J, Henney A: Matrix metalloproteinases and cardiovascular disease. Circ Res 1995, 77:863-868[Free Full Text]
3. Davies M, Richardson P, Woolf N, Katz D, Mann J: Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage, and smooth muscle cells content. Br Heart J 1993, 69:377-381[Abstract/Free Full Text]
4. Lendon CL, Davies MJ, Born GV, Richardson PD: Atherosclerotic plaque caps are locally weakened when macrophages density is increased. Atherosclerosis 1991, 87:87-90[Medline]
5. Nikkari S, O'Brien K, Ferguson M, Hatsukami T, Welgus H, Clowes A: Interstitial collagenase (MMP-1) expression in human carotid atherosclerotics. Circulation 1995, 92:1393-1398[Abstract/Free Full Text]
6. Galis Z, Sukhova G, Lark M, Libby P: Increased expression of matrix metalloproteinases and matrix degradation activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994, 94:2493-2503
7. Goetzl E, Banda M, Leppert D: Matrix metalloproteinases in immunity. J Immunol 1996, 156:1-4[Abstract]
8. Welgus H, Campbell E, Cury J, Eisen A, Senior RM, Goldberg G: Neutral metalloproteinases produced by human mononuclear phagocytes: enzyme profile, regulation, and expression during cellular development. J Clin Invest 1990, 86:1496-1502
9. Saren P, Welgus H, Kovanen P: TNF- and IL-1ß selectively induce expression of 92-kd gelatinase by human macrophages. J Immunol 1996, 157:4159-4165[Abstract]
10. Schoenbeck U, Mach F, Sukhova G, Murphy C, Bonnefoy J-Y, Fabunmi R, Libby P: Regulation of matrix metalloproteinase expression in human vascular smooth muscle cells by T lymphocytes. Circ Res 1997, 81:448-454[Abstract/Free Full Text]
11. Schroen DJ, Brinckerhoff C: Nuclear hormone receptors inhibit matrix metalloproteinases (MMP) gene expression through diverse mechanisms. Gene Exp 1996, 6:197-207
12. Schoonjans K, Martin G, Staels B, Auwerx J: Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol 1997, 8:159-166[Medline]
13. Vidal-Puig AJ, Considine RV, Jimenez-Linan MA, Pories WJ, Caro JF, Flier JS: Peroxisome proliferator-activated receptor gene expression in human tissues: effects of obesity, weight loss, and regulation by insulin and glucocorticoids. J Clin Invest 1997, 99:2416-2422[Medline]
14. Tontonoz P, Hu E, Spiegelman BM: Stimulation of adipogenesis in fibroblasts by PPAR 2, a lipid-activated transcription factor. Cell 1994, 79:1147-1156[Medline]
15. Brun R, Tontonoz P, Forman B, Ellis RJ, Evan R, Spiegelman BM: Differential activation of adipogenesis by multiple PPAR isoforms. Genes Dev 1996, 10:974-984[Abstract/Free Full Text]
16. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans R: The nuclear receptor superfamily: the second decade. Cell 1995, 83:835-839[Medline]
17. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans R: M. 15-Deoxy-delta 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR . Cell 1995, 83:803-812[Medline]
18. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris D, Lehmann JM: A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor and promotes adipocyte differentiation. Cell 1995, 83:813-819[Medline]
19. Yu K, Bayona W, Kallen CB, Harding HP, Ravera CP, McMahon G, Brown M, Lazar MA: Differential activation of peroxisome proliferator-activated receptors by eicosanoids. J Biol Chem 1995, 270:23975-23983[Abstract/Free Full Text]
20. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA: An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor . J Biol Chem 1995, 270:12953-12956[Abstract/Free Full Text]
21. Willson TM, Cobb JE, Cowan DJ, Wiethe RW, Correa ID, Prakash SR, Beck KD, Moore LB, Kliewer SA, Lehmann JM: The structure-activity relationship between peroxisome proliferator-activated receptor agonism and the antihyperglycemic activity of thiazolidinediones. J Med Chem 1996, 39:665-668[Medline]
22. Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB, Koble CS, Devchand P, Wahli W, Willson TM, Lenhard JM, Lehmann JM: Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors and . Proc Natl Acad Sci USA 1997, 94:4318-4323[Abstract/Free Full Text]
23. Greene M, Blumberg B, McBride O, Kronquist K, Kwan K, Hsieh L, Greene G, Nimer S: Isolation of the human peroxisome proliferator activated receptor cDNA: expression in hematopoietic cells and chromosomal mapping. Gene Expression 1995, 4:281-299[Medline]
24. Jiang C, Ting A, Seed B: PPAR- agonists inhibit production of monocyte inflammatory cytokines. Nature 1998, 391:82-86[Medline]
25. Ricote M, Li A, Wilson T, Kelly C, Glass C: The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature 1998, 391:79-82[Medline]
26. Ross R: The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993, 362:801-809[Medline]
27. Raines E, Ross R: Is overamplification of the normal macrophage defensive role critical to lesion development? Ann NY Acad Sci 1997, 811:76-85[Free Full Text]
28. Galis Z, Sukhova G, Kranzhoefer R, Clark S, Libby P: Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases. Proc Natl Acad Sci USA 1995, 92:402-406[Abstract/Free Full Text]
29. Henney A, Wakeley P, Davies M, Foster K, Hembry R, Murphy G, Humphries S: Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci USA 1991, 88:8154-8158[Abstract/Free Full Text]
30. Galis Z, Sukhova G, Libby P: Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue. FASEB J 1995, 9:974-980[Abstract]
31. Fukushima M: Biological activities and mechanisms of action of PGJ₂ and related compounds: an update. Prostaglandins Leukotrienes Essent Fatty Acids 1992, 47:1-12[Medline]
32. Giles H, Leff P: The biology and pharmacology of PGD₂. Prostaglandins 1988, 35:277-300[Medline]
33. Urade Y, Ujihara M, Horigushi Y, Ikai K, Hayaishi O: The major source of endogenous prostaglandin D2 is likely antigen-presenting cells. J Immunol 1989, 143:2982-2989[Abstract]
34. Nolen J, Ludvik B, Beerdsen P: Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N Engl J Med 1994, 331:1188-1193[Abstract/Free Full Text]
35. Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, Shulman GI: Efficacy and metabolic effects of merformin and troglitazone in type II diabetes mellitus. N Engl J Med 1998, 338:867-872[Abstract/Free Full Text]
36. Schwartz S, Raskin P, Fonseca V, Graveline JF: Effect of troglitazone in insulin-treated patients with type II diabetes mellitus. N Engl J Med 1998, 338:861-866[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Collino, N. S.A. Patel, and C. Thiemermann Review: PPARs as new therapeutic targets for the treatment of cerebral ischemia/reperfusion injury Therapeutic Advances in Cardiovascular Disease, June 1, 2008; 2(3): 179 - 197. [Abstract] [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]

				E. Rigamonti, C. Fontaine, B. Lefebvre, C. Duhem, P. Lefebvre, N. Marx, B. Staels, and G. Chinetti-Gbaguidi Induction of CXCR2 Receptor by Peroxisome Proliferator-Activated Receptor {gamma} in Human Macrophages Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 932 - 939. [Abstract] [Full Text] [PDF]

				S. Z. Duan, M. G. Usher, and R. M. Mortensen Peroxisome Proliferator-Activated Receptor-{gamma}-Mediated Effects in the Vasculature Circ. Res., February 15, 2008; 102(3): 283 - 294. [Abstract] [Full Text] [PDF]

				D. K. McGuire and S. E. Inzucchi New Drugs for the Treatment of Diabetes Mellitus: Part I: Thiazolidinediones and Their Evolving Cardiovascular Implications Circulation, January 22, 2008; 117(3): 440 - 449. [Full Text] [PDF]

				A. S. Kelly, A. M. Thelen, D. R. Kaiser, J. M. Gonzalez-Campoy, and A. J. Bank Rosiglitazone improves endothelial function and inflammation but not asymmetric dimethylarginine or oxidative stress in patients with type 2 diabetes mellitus Vascular Medicine, November 1, 2007; 12(4): 311 - 318. [Abstract] [PDF]

				C. Schindler Review: The metabolic syndrome as an endocrine disease: is there an effective pharmacotherapeutic strategy optimally targeting the pathogenesis? Therapeutic Advances in Cardiovascular Disease, October 1, 2007; 1(1): 7 - 26. [Abstract] [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]

				F. Demirturk, H. Aytan, A. Caliskan, P. Aytan, T. Yener, D. Koseoglu, and A. Yenisehirli The effect of rosiglitazone in the prevention of intra-abdominal adhesion formation in a rat uterine horn model Hum. Reprod., November 1, 2006; 21(11): 3008 - 3013. [Abstract] [Full Text] [PDF]

				Z. T. Bloomgarden Third Annual World Congress on the Insulin Resistance Syndrome: Atherothrombotic disease Diabetes Care, August 1, 2006; 29(8): 1973 - 1980. [Full Text] [PDF]

				K. Wang, Z. Zhou, M. Zhang, L. Fan, F. Forudi, X. Zhou, W. Qu, A. M. Lincoff, A. M. Schmidt, E. J. Topol, et al. Peroxisome Proliferator-Activated Receptor {gamma} Down-Regulates Receptor for Advanced Glycation End Products and Inhibits Smooth Muscle Cell Proliferation in a Diabetic and Nondiabetic Rat Carotid Artery Injury Model J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 37 - 43. [Abstract] [Full Text] [PDF]

				J. Banerjee and C. M Komar Effects of luteinizing hormone on peroxisome proliferator-activated receptor {gamma} in the rat ovary before and after the gonadotropin surge Reproduction, January 1, 2006; 131(1): 93 - 101. [Abstract] [Full Text] [PDF]

				S Soumian, R Gibbs, A Davies, and C Albrecht mRNA expression of genes involved in lipid efflux and matrix degradation in occlusive and ectatic atherosclerotic disease J. Clin. Pathol., December 1, 2005; 58(12): 1255 - 1260. [Abstract] [Full Text] [PDF]

				M. Xia, M. Hou, H. Zhu, J. Ma, Z. Tang, Q. Wang, Y. Li, D. Chi, X. Yu, T. Zhao, et al. Anthocyanins Induce Cholesterol Efflux from Mouse Peritoneal Macrophages: THE ROLE OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {gamma}-LIVER X RECEPTOR {alpha}-ABCA1 PATHWAY J. Biol. Chem., November 4, 2005; 280(44): 36792 - 36801. [Abstract] [Full Text] [PDF]

				S. H. Han, M. J. Quon, and K. K. Koh Beneficial Vascular and Metabolic Effects of Peroxisome Proliferator-Activated Receptor-{alpha} Activators Hypertension, November 1, 2005; 46(5): 1086 - 1092. [Abstract] [Full Text] [PDF]

				L. Klotz, M. Schmidt, T. Giese, M. Sastre, P. Knolle, T. Klockgether, and M. T. Heneka Proinflammatory Stimulation and Pioglitazone Treatment Regulate Peroxisome Proliferator-Activated Receptor {gamma} Levels in Peripheral Blood Mononuclear Cells from Healthy Controls and Multiple Sclerosis Patients J. Immunol., October 15, 2005; 175(8): 4948 - 4955. [Abstract] [Full Text] [PDF]

				T. J. Standiford, V. G. Keshamouni, and R. C. Reddy Peroxisome Proliferator-activated Receptor-{gamma} as a Regulator of Lung Inflammation and Repair Proceedings of the ATS, October 1, 2005; 2(3): 226 - 231. [Abstract] [Full Text] [PDF]

				N. Hennuyer, A. Tailleux, G. Torpier, H. Mezdour, J.-C. Fruchart, B. Staels, and C. Fievet PPAR{alpha}, but not PPAR{gamma}, Activators Decrease Macrophage-Laden Atherosclerotic Lesions in a Nondiabetic Mouse Model of Mixed Dyslipidemia Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1897 - 1902. [Abstract] [Full Text] [PDF]

				A. Pfutzner, N. Marx, G. Lubben, M. Langenfeld, D. Walcher, T. Konrad, and T. Forst Improvement of Cardiovascular Risk Markers by Pioglitazone Is Independent From Glycemic Control: Results From the Pioneer Study J. Am. Coll. Cardiol., June 21, 2005; 45(12): 1925 - 1931. [Abstract] [Full Text] [PDF]

				S Soumian, C Albrecht, A. Davies, and R. Gibbs ABCA1 and atherosclerosis Vascular Medicine, May 1, 2005; 10(2): 109 - 119. [Abstract] [PDF]

				N. P. Kadoglou, S. S. Daskalopoulou, D. Perrea, and C. D. Liapis Matrix Metalloproteinases and Diabetic Vascular Complications Angiology, March 1, 2005; 56(2): 173 - 189. [Abstract] [PDF]

				M. Abdelrahman, A. Sivarajah, and C. Thiemermann Beneficial effects of PPAR-{gamma} ligands in ischemia-reperfusion injury, inflammation and shock Cardiovasc Res, March 1, 2005; 65(4): 772 - 781. [Abstract] [Full Text] [PDF]

				C. J. Scotton, F. O. Martinez, M. J. Smelt, M. Sironi, M. Locati, A. Mantovani, and S. Sozzani Transcriptional Profiling Reveals Complex Regulation of the Monocyte IL-1{beta} System by IL-13 J. Immunol., January 15, 2005; 174(2): 834 - 845. [Abstract] [Full Text] [PDF]

				S. C. Tyagi, W. Rodriguez, A. M. Patel, A. M. Roberts, J. C. Falcone, J. C. Passmore, J. T. Fleming, and I. G. Joshua Hyperhomocysteinemic Diabetic Cardiomyopathy: Oxidative Stress, Remodeling, and Endothelial-Myocyte Uncoupling Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 2005; 10(1): 1 - 10. [Abstract] [PDF]

				A. C. Newby Dual Role of Matrix Metalloproteinases (Matrixins) in Intimal Thickening and Atherosclerotic Plaque Rupture Physiol Rev, January 1, 2005; 85(1): 1 - 31. [Abstract] [Full Text] [PDF]

				S. Giri, R. Rattan, A. K. Singh, and I. Singh The 15-Deoxy-{delta}12,14-Prostaglandin J2 Inhibits the Inflammatory Response in Primary Rat Astrocytes via Down-Regulating Multiple Steps in Phosphatidylinositol 3-Kinase-Akt-NF-{kappa}B-p300 Pathway Independent of Peroxisome Proliferator-Activated Receptor {gamma} J. Immunol., October 15, 2004; 173(8): 5196 - 5208. [Abstract] [Full Text] [PDF]

				X. Wang, J. Liang, T. Koike, H. Sun, T. Ichikawa, S. Kitajima, M. Morimoto, H. Shikama, T. Watanabe, Y. Sasaguri, et al. Overexpression of Human Matrix Metalloproteinase-12 Enhances the Development of Inflammatory Arthritis in Transgenic Rabbits Am. J. Pathol., October 1, 2004; 165(4): 1375 - 1383. [Abstract] [Full Text] [PDF]

				M. Francois, P. Richette, L. Tsagris, M. Raymondjean, M.-C. Fulchignoni-Lataud, C. Forest, J.-F. Savouret, and M.-T. Corvol Peroxisome Proliferator-activated Receptor-{gamma} Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element J. Biol. Chem., July 2, 2004; 279(27): 28411 - 28418. [Abstract] [Full Text] [PDF]