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(Circulation. 2000;101:1311.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Expression and Function of PPAR in Rat and Human Vascular Smooth Muscle Cells

Ronald E. Law, PhD; Stephan Goetze, MD; Xiao-Ping Xi, MD; Simon Jackson, PhD; Yasuko Kawano, MD; Linda Demer, MD, PhD; Michael C. Fishbein, MD; Woerner P. Meehan, PhD; Willa A. Hsueh, MD

From the Department of Medicine (R.E.L., S.G., X.-P.X., S.J., Y.K., L.D., W.P.M., W.A.H.); the Division of Endocrinology, Diabetes, and Hypertension (R.E.L., S.G., X.-P.X., Y.K., W.P.M., W.A.H.); the Division of Cardiology (S.J., L.D.); and the Department of Pathology and Laboratory Medicine (M.C.F.), University of California at Los Angeles School of Medicine.

Correspondence to Ronald E. Law, PhD, UCLA, Warren Hall, Second Floor, Suite 24-130, 900 Veteran Ave, Los Angeles, CA 90095.

	Abstract

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Background—Peroxisome proliferator–activated receptor- (PPAR) is activated by fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZDs). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell (VSMC) proliferation and migration in vitro and in postinjury intimal hyperplasia.

Methods and Results—Rat and human VSMCs express mRNA and nuclear receptors for PPAR1. Three PPAR ligands, the TZDs TRO and rosiglitazone and the prostanoid 15-deoxy-^12,14-prostaglandin J2 (15d-PGJ2), all inhibited VSMC proliferation and migration. PPAR is upregulated in rat neointima at 7 days and 14 days after balloon injury and is also present in early human atheroma and precursor lesions.

Conclusions—Pharmacological activation of PPAR expressed in VSMCs inhibits their proliferation and migration, potentially limiting restenosis and atherosclerosis. These receptors are upregulated during vascular injury.

Key Words: atherosclerosis • restenosis • growth substances • migration • thiazolidinediones

	Introduction

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Peroxisome proliferator–activated receptor- (PPAR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPAR expression is abundant in adipose tissue, where it promotes adipocyte differentiation and regulates expression of genes involved in fatty acid metabolism.¹ Various fatty acids and eicosanoids are likely physiological ligands for PPAR.^{2 3 4} Thiazolidinediones (TZDs) are oral antidiabetic compounds that enhance sensitivity to the metabolic effects of insulin and that bind with high affinity to PPAR.⁵ In humans and animals with insulin resistance and type 2 diabetes, TZDs ameliorate hyperglycemia, hyperinsulinemia, and hypertriglyceridemia.^{6 7 8}

We previously reported that troglitazone (TRO) suppressed neointima formation in rat aorta after endothelial injury, most likely as a result of direct vascular action to inhibit vascular smooth muscle cell (VSMC) growth and migration.⁹ However, our initial studies did not address whether the vascular effects of TRO were mediated through PPAR, which at that time was thought to be highly restricted to adipose tissue. Recent studies have identified PPAR in a variety of nonadipose tissues: skeletal muscle,^{10 11} heart,¹¹ kidney proximal tubules,¹² colon,¹³ bone marrow stromal cells,¹⁴ neutrophils,¹⁴ macrophages,^{15 16 17 18 19 20} and breast carcinoma,²¹ which implicates novel functions for this receptor distinct from its well-characterized metabolic activity. TRO, however, is distinguishable from other TZD PPAR ligands because it also contains a vitamin E moiety, which is also known to inhibit VSMC growth and intimal hyperplasia.²² The vascular effects of TRO, therefore, could be independent of PPAR.

The expression and function of PPAR in VSMCs is somewhat controversial. In human VSMCs, Staels et al²³ observed faint expression of PPAR that was not involved in the negative regulation of cytokine-induced interleukin-6 and cyclooxygenase-2 expression, this effect being mediated by PPAR. In contrast, a recent study reported that human VSMCs express PPAR, which inhibited matrix metalloproteinase expression and cell migration.²⁴ Therefore, we examined the expression and function of PPAR in rat and human VSMCs, focusing on VSMC growth and migration.

	Results

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PPAR Ligands Inhibit bFGF-Induced DNA Synthesis in Rat and Human VSMCs
The TZD PPAR ligands TRO and RSG, and 15-deoxy-^12,14-prostaglandin J2 (15d-PGJ2), a non-TZD PPAR ligand, all inhibited basic fibroblast growth factor (bFGF)-induced DNA synthesis in rat VSMCs (Figure 1< pick;3702f1;0;;;ZPICKFOOT;F1>). At concentrations of 5 µmol/L, rosiglitazone (RSG) and TRO inhibited DNA synthesis by 65% and 58%, respectively. 15d-PGJ2 was a far more potent inhibitor of VSMC DNA synthesis, reducing [³H]thymidine incorporation by 64.2±7% and 95±3.4% at 0.1 and 1 µmol/L, respectively.

View larger version (44K): [in this window] [in a new window]   Figure 1. PPAR ligands inhibit bFGF-stimulated DNA synthesis in rat aortic VSMCs. DNA synthesis was measured in quiescent cells stimulated with 20 ng/mL bFGF for 24 hours as detailed in Methods. Values are expressed as mean±SEM (n=4; compared with bFGF alone: #P<0.05, @P<0.01, *P<0.001).

In human coronary artery VSMCs (CASMCs), all tested PPAR ligands inhibited bFGF-stimulated DNA synthesis (Figure 2). RSG and 15d-PGJ2 were more potent than TRO. Even at 0.1 µmol/L, RSG and 15d-PGJ2 caused a statistically significant inhibition of CASMC proliferation (25.5±5.5% and 42.8±5.8%, respectively). Inhibition of 50% was observed at concentrations >0.5 µmol/L for 15d-PGJ2 or >1 µmol/L for RSG. TRO exhibited weaker antiproliferative activity with a maximum effect of 45.5±4.7% inhibition observed at 10 µmol/L.

View larger version (49K): [in this window] [in a new window]   Figure 2. PPAR ligands inhibit bFGF-induced DNA synthesis in human coronary artery VSMCs. Treatment of cells and measurement of DNA synthesis were performed as described in Figure 1. Values are expressed as mean±SEM (n=4; compared with bFGF alone: #P<0.05, @P<0.01, *P<0.001).

TRO and RSG had no effect on VSMC viability at 10 µmol/L, but 5 µmol/L 15d-PGJ2 for 48 hours induced significant cell death.

PPAR Ligands Inhibit PDGF-Directed Migration in Rat and Human VSMCs
Platelet-derived growth factor (PDGF) is one of the most potent in vitro chemoattractants for VSMCs. TRO, RSG, and 15d-PGJ2 all blocked PDGF-directed VSMC migration (Figure 3). PDGF induced a 5.6-fold increase in the number of rat VSMCs that migrated through the gelatin-coated membrane. TRO and RSG inhibited PDGF-directed migration in a dose-dependent manner at concentrations of 0.1 to 10 µmol/L. A modest but statistically significant effect was observed at 0.1 µmol/L: At 1 µmol/L for RSG and at 5 µmol/L for TRO, migration was inhibited by >50%. At 10 µmol/L, RSG totally abolished PDGF-directed migration.

View larger version (39K): [in this window] [in a new window]   Figure 3. PPAR ligands inhibit PDGF-directed migration of rat aortic VSMCs. Migration was assayed 4 hours after addition of PDGF-BB (20 ng/mL) and is expressed as x-fold over control (control, 5.0±0.4 cells per field; PDGF, 28.2±2.8 cells per field). For each experiment, 8 to 12 fields were counted. Data are expressed as mean±SEM (n=6; compared with PDGF alone: #P<0.05, @P<0.01, *P<0.001).

In contrast to its strong antiproliferative activity, 15d-PGJ2 was only a slightly more potent inhibitor of PDGF-directed migration than TRO or RSG. The concentrations of TRO, RSG, and 15d-PGJ2 required to inhibit PDGF-directed migration by 50% were 2.4 µmol/L, 0.3 µmol/L, and 0.2 µmol/L, respectively. TRO, RSG, and 15d-PGJ2 also inhibited PDGF-directed migration of human CASMCs with very similar dose-response curves (Figure 4).

View larger version (34K): [in this window] [in a new window]   Figure 4. PPAR ligands inhibit PDGF-directed migration of human CASMCs. Migration was assayed as described for Figure 3 (control, 4.2±0.6 cells per field; PDGF, 24.3±1.2 cells per field). Data are expressed as mean±SEM (n=4; compared with PDGF alone: #P<0.05, *P<0.001).

Rat and Human VSMCs Express PPAR mRNA
The PPAR gene produces 2 major mRNA species through alternative promoter usage.¹¹ Adipose tissues express both isoforms, but PPAR1 expression is much higher than PPAR2 in nonadipose tissues.^{10 11} Using a sensitive RNase protection assay (RPA) (Figure 5), we observed only faint expression of PPAR1 mRNA in mouse 3T3-L1 preadipocyte cells, whereas significant upregulation of both PPAR1 and -2 mRNAs occurred during their in vitro differentiation to adipocytes.³ VSMCs from rat aorta and human VSMCs from umbilical artery, coronary artery, and aorta expressed PPAR1 mRNA exclusively, as evidenced by the single protected band of 258 (human) or 185 (rat) bases. Although rat aortic tissue contained PPAR1 and -2, the presence of PPAR2 mRNA in aorta and its absence in cultured VSMCs are most likely due to contaminating adventitial fat. Human umbilical vein endothelial cells also prominently expressed PPAR1 but not -2 mRNA.

View larger version (40K): [in this window] [in a new window]   Figure 5. Rat and human VSMCs express PPAR mRNA. RPA was performed on 10 µg of total RNA. Yeast RNA (10 µg) was used as a negative control. GAPDH was assayed separately to verify integrity of input RNA. AOSMC indicates rat or human aortic VSMCs; UASMC, human umbilical artery VSMCs; HUVEC, human umbilical vein endothelial cells; and AORTA, aorta from uninjured Sprague-Dawley rat. Data are representative of 3 separate RNA preparations from cultured cells or aortas.

Expression and Subcellular Localization of PPAR in Rat and Human VSMCs
To detect PPAR protein in VSMCs, we performed Western immunoblotting using a murine monoclonal antibody to human recombinant PPAR (Glaxo Wellcome) previously shown to recognize 2 bands of 56 and 52 kDa, corresponding to PPAR2 and -1, respectively, in 3T3-L1 adipocyte nuclear extracts (Figure 6).²⁵ Receptor levels were low in nuclear extracts of undifferentiated 3T3-L1 preadipocytes. Cultured aortic and human coronary artery VSMCs expressed only PPAR1, which was present almost exclusively in the nuclear fraction (Figure 6). Nuclear extracts from rat and human VSMCs contain a protein with a molecular weight greater than that of PPAR2 that is probably not PPAR2, because these cells do not express detectable mRNA for this isoform by RPA (see Figure 5).

View larger version (30K): [in this window] [in a new window]   Figure 6. Rat and human VSMCs express PPAR protein. Nuclear extract (NE; 25 µg) from undifferentiated 3T3-L1 (PRE-ADIP) or differentiated 3T3-L1 adipocytes (ADIP), 25 µg of NE or cytosolic proteins of rat aortic VSMCs and human CASMCs, or 50 µg of total protein from an uninjured rat aorta were assayed by Western immunoblotting. Arrows and lines denote expected positions for PPAR1 and -2 with differentiated 3T3-L1 adipocytes used as a positive control (* indicates artifact band). Data are representative of 3 nuclear extract preparations.

Whole-tissue extracts from normal rat aortas contained PPAR1 and -2 protein, consistent with the pattern of PPAR mRNA expression detected by RPA (Figure 5).

PPAR Expression in Human Vascular Lesions and Rat Neointima
In human atherosclerotic lesions, PPAR is expressed in macrophages and to a lesser extent in VSMCs.^{19 20} To validate the quality of PPAR antibodies used for immunohistochemical analysis, we first examined human coronary arteries for receptor expression. Immunoreactive PPAR colocalizes with macrophages visualized by staining of parallel sections of a type II atherosclerotic lesion (Figure 7)²⁶ with the macrophage-specific antibody anti-CD68. In a type I lesion exhibiting adaptive intimal thickening, faint expression of PPAR is seen both in neointimal regions devoid of CD68-positive cells and in the underlying media in VSMCs, as demonstrated in serial sections stained with antibody against -smooth muscle actin. Similar results were obtained with either of the 2 commercial antibodies to stain 2 additional type I and type II lesions from separate biopsies.

View larger version (81K): [in this window] [in a new window]   Figure 7. Expression of PPAR in early-stage human atheroma (type II) (a) and a precursor lesion (d) with adaptive intimal thickening (type I). High-power views show immunoreactive PPAR (detected with a polyclonal rabbit anti-human PPAR antibody from Biomol) in medial and intimal VSMCs (I indicates intima; M, media). In type II (early atheroma) lesions, highest levels of immunoreactive PPAR colocalized with macrophages detected in parallel sections stained with macrophage marker CD68 (b, e). Staining for -smooth muscle actin was used as a marker for VSMCs (c, f). Data are representative of 3 type I and type II lesions examined.

In neointima formed after balloon injury of rat aortas, faint expression of PPAR is observed in the media of uninjured vessels (Figure 8). Neointima that developed at 7 and 14 days after balloon injury displayed intense staining for immunoreactive PPAR, which suggests that this receptor is upregulated in response to vascular injury. VSMCs were the major cell type present in rat neointima, as shown by its strong positive staining for -smooth muscle actin and the absence of staining for the macrophage marker ED1. Immunoreactive PPAR did not localize specifically to the nucleus of neointimal or medial VSMCs, because staining of the cytoplasm was observed. We do not know whether VSMCs in arterial vessels actually contain PPAR in their cytoplasm or whether this is an artifact of tissue fixation.

View larger version (96K): [in this window] [in a new window]   Figure 8. Expression of PPAR in intimal VSMCs after balloon injury of rat aortas. High-power view shows strong immunoreactive staining for PPAR in neointima at 7 and 14 days after balloon injury (c, d) (I indicates intima; M, media). Faint staining for PPAR is observed in media of an uninjured control aorta (a) and at 2 days after injury (b). Few macrophages were present in a 14-day lesion (e), because there was no staining for macrophage-specific marker ED1 (Serotec Laboratories). VSMCs in a 14-day lesion were detected by immunostaining for -smooth muscle actin (f). Data are representative of 3 aortas examined per time point.

To confirm that the immunoreactive signal detected in rat neointima was bona fide PPAR, we used nuclear extracts from differentiated 3T3-L1 adipocytes to preabsorb PPAR antibodies before their use in immunostaining. Addition of 50 µg of nuclear extracts of differentiated 3T3-L1 adipocytes, which contain high levels of PPAR1 and -2 compared with undifferentiated 3T3-L1 cells (see Figure 6), markedly attenuated staining in both the neointima and media (Figure 9), whereas extracts from undifferentiated 3T3-L1 preadipocytes had little effect. Thus, the immunoreactivity observed in these tissues corresponds to PPAR protein.

View larger version (108K): [in this window] [in a new window]   Figure 9. Nuclear extracts from 3T3-L1 adipocytes block immunostaining of 14-day neointima by PPAR antibody. Preabsorption of polyclonal antibody to human PPAR with nuclear extracts from 3T3-L1 adipocytes substantially diminishes immunostaining in 14-day neointima (compare b vs c) (I indicates intima; M, media), whereas extract from undifferentiated 3T3-L1 cells lacked this effect (compare b vs d). Negative control (a) was stained with nonspecific rabbit IgG. Data are representative of 3 preabsorption experiments.

	Discussion

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PPAR mRNA and protein have previously been identified in rat aortic and human saphenous vein VSMCs.^{24 27} In human aortic VSMCs, Staels et al²³ found extremely low levels of PPAR mRNA relative to PPAR message detected by RPA. However, the pattern of PPAR isoform expression was not described. Using an RPA that permits the detection of both isoforms, we found that PPAR1 mRNA is easily detectable in cultured rat aortic VSMCs, in whole aortas from uninjured animals, and in human VSMCs. PPAR1 expression was substantially higher in VSMCs from human coronary artery and umbilical artery than in cells from aortas. None of these cells expressed detectable PPAR2 mRNA.

Cultured VSMCs from rat aortas and human coronary arteries expressed only PPAR1 protein, consistent with their pattern of mRNA isoform expression. Rat aortic tissue contained receptors for both PPAR1 and -2, consistent with the presence of both mRNA species in that tissue. Levels of PPAR1 protein in rat aortic and human coronary arterial VSMCs appeared to be substantial, because they express levels similar to those of differentiated 3T3-L1 adipocytes, a major in vitro model for studying PPAR function. Our data are also consistent with previous studies showing that PPAR is present in rat aortic and human saphenous vein VSMCs.^{24 27}

Inhibition of VSMC growth and migration in vitro occurred at low micromolar concentrations of PPAR ligands, which are achievable in the circulation of humans or animals given TRO for insulin sensitization.²⁸ TRO and RSG had comparable activities to inhibit VSMC growth and migration. This finding is somewhat surprising, because other studies have shown RSG to be 5- to 20-fold more efficacious than TRO in binding to PPAR and in increasing transcriptional activity of PPAR,² stimulating insulin-mediated glucose transport,⁴ lowering hyperglycemia in ob/ob diabetic mice,⁴ and inducing adipocyte differentiation.³ The roughly equal potencies between TRO and RSG for inhibiting VSMC proliferation and migration may be the result of TRO being a bifunctional molecule having both a TZD and -tocopherol (vitamin E) moiety. Tocopherol inhibits VSMC proliferation and macrophage migration.^{22 29} TRO has also recently been shown to inhibit cholesterol synthesis through a mechanism independent of its vitamin E or PPAR ligand properties.³⁰ The vascular effects of TRO, therefore, may be complex, with its activity mediated partially through -tocopherol and/or other PPAR-independent mechanisms and partially through PPAR. RSG lacks -tocopherol and is a more "pure" PPAR ligand. Its vascular effects are likely to be mediated exclusively through PPAR. The non-TZD PPAR ligand 15d-PGJ2 displayed the strongest antiproliferative and antimigration activity in VSMCs. RSG is 20-fold more potent than 15d-PGJ2 in activating PPAR as a transcription factor in transient transfection experiments and in inducing differentiation of 3T3-L1 cells into adipocytes.^{2 3} The biological effects of 15d-PGJ2, however, are complex because of its potential to activate prostaglandin receptors. RSG, therefore, may provide the clearest evidence for PPAR-mediated effects. The vascular effects of RSG we observed importantly distinguish this study from that of Marx et al,²⁴ which used only TRO and 15d-PGJ2 to inhibit human VSMC migration.

The molecular basis for the inhibition of VSMC growth and migration by PPAR remains to be elucidated. PPAR-mediated inhibition of transcription factor function (ie, transrepression) critical for these processes is probably involved. We previously observed that TRO inhibited the activity of ELK-1, an ets-family transcription factor, after mitogenic stimulation of VSMCs by bFGF.⁹ Transrepression of ELK-1, and possibly other transcription factors, by TRO may be the underlying mechanism for its inhibition of VSMC growth and migration and hence intimal hyperplasia.⁹ This hypothesis is supported by studies in macrophages in which PPAR also negatively regulates gene expression.^{16 17} Iijima et al²⁷ observed that TRO and 15d-PGJ2 poorly activate (<1-fold induction) endogenous PPAR in rat VSMCs, a finding we reproduced (unpublished data). By comparison, PPAR present in 3T3-L1 adipocytes or overexpressed by transfection in CV-1 renal fibroblasts show a 5- to 100-fold increase in transcription factor activity in response to RSG, TRO, or 15d-PGJ2.^{2 3 5} These data suggest that transcriptional activation by PPAR may have a different pharmacology than transrepression by these receptors and is dependent on the cell type.

To date, only 2 previous studies have described the expression of PPAR in normal or diseased vasculature. Immunohistochemical analysis of PPAR human atherosclerotic lesions revealed strong expression in macrophages, with fainter expression observed in VSMCs. VSMCs in the underlying media of lesions or in unaffected areas of the coronary artery had nearly undetectable levels of PPAR.^{19 20} In early human atheroma (type II), we found that the highest levels of PPAR colocalized with macrophages in the neointima. VSMCs present in the neointima and the underlying media stained positively for PPAR, but staining was less than in macrophages. We also observed significant staining for PPAR in human VSMCs present in regions of adaptive intimal thickening in type I lesions that can be precursors to atheromas.

Our study also provides new insight concerning the in vivo expression of PPAR in the injured vasculature. Neointimal VSMCs prominently upregulate PPAR protein levels. Lesions that result from this model of vascular injury differ from atheromas in several important aspects. First, intimal hyperplasia after mechanical injury is a more acute response than atherosclerosis, which develops over a longer period of time. Second, VSMCs are the predominant cell type in balloon injury–induced neointimal lesions, where we find little infiltration of macrophages. In contrast, macrophages are abundant in atherosclerotic lesions and play a major role in driving atherogenesis.³¹ Therefore, upregulation of PPAR and its activation by physiological or pharmacological ligands in the damaged vasculature may be important in limiting lesions dependent on VSMC activity.

The present data are in stark contrast to a recent report emphasizing the role of PPAR and dismissing involvement of PPAR in VSMC responses that promote restenosis and atherosclerosis. In that study, Staels et al,²³ using a different antibody and not using nuclear extracts, did not detect significant levels of PPAR in human aortic VSMCs. Using RPA, we find that human aortic VSMCs express much lower levels of PPAR mRNA than human coronary VSMCs. Either or both of these differences may have resulted in our experimental approach being more sensitive for detecting PPAR protein. We also found that PPAR ligands had no effect on VSMC inflammatory responses, whereas we find that PPAR ligands have antiproliferative and antimigratory activity in VSMCs.

The present results have important implications for diabetes-associated vascular disease. In type 2 diabetes, the development of both atherosclerosis and restenosis is substantially accelerated.³² We and others have suggested that TZD may retard atherogenesis and restenosis through their inhibitory effects on VSMCs^{9 24} and macrophages^{17 19 20} in the damaged vasculature. TZDs, therefore, may provide a dual benefit for type 2 diabetes by ameliorating insulin resistance and its metabolic sequelae, as well as directly protecting the vasculature from injury.

	Methods

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Materials
TRO was kindly provided by Parke Davis; rosiglitazone was a generous gift from SmithKline Beecham. All other materials were purchased from commercial suppliers.

Cell Culture and Treatment With Growth Factors and PPAR Ligands
Rat aortic VSMCs were prepared from thoracic aorta of 2- to 3-month-old Sprague-Dawley rats (Charles River) and assessed for purity as previously described.¹³ Human vascular cells were purchased from Clonetics and cultured as recommended.Subconfluent VSMCs (passage 5 or less) were made quiescent by serum starvation (0.4% FBS). PPAR ligands were added 30 minutes before growth factors. Independent preparations of VSMCs were used for each experiment in the n value.

DNA Synthesis
Quiescent VSMCs were stimulated with 20 ng/mL basic fibroblast growth factor (bFGF) in the presence or absence of PPAR ligands for 48 hours. During the final 6 hours of the incubation, cells were pulsed with 1 µCi of [³H]thymidine/mL. Trichloroacetic acid–precipitable [³H]thymidine incorporation measurements are the average of triplicate wells.

Migration
VSMC migration was examined in transwell cell culture chambers with a gelatin-coated polycarbonate membrane with 8-µm pores as previously published.⁹

RNase Protection Assay
RNase protection assays (RPAs) were performed with antisense RNA probes prepared from PPAR cDNA (kindly provided by Dr J. Flier, Harvard University, Boston, Mass) as described in Reference ¹¹ . RPA detected protected bands of rat PPAR1, 185 bases, rat PPAR2, 273 bases, human PPAR1, 258 bases, human PPAR2, 348 bases, rat GAPDH, 97 bases, and human GAPDH, 96 bases.

Immunoblotting
Protein extracts from aortas were prepared by removal of adventitial fat, homogenization after quick-freezing in liquid nitrogen, and centrifugation to remove debris.

Nuclear and cytosolic fractions from cultured VSMCs were prepared by the method of Dignam et al.³³ Equal amounts of proteins (25 to 50 µg) were electrophoresed and transferred to nitrocellulose membranes. Membranes were incubated with anti-PPAR antibodies, either a murine monoclonal (Glaxo-Wellcome) or rabbit polyclonal (Biomol or Santa Cruz) at a concentration of 1:1000 for 2 hours in 0.2 mol/L Tris-HCl pH 7.5, 0.5 mol/L NaCl buffer containing 5% fat-free milk powder and 0.1% Tween 20. Blots were washed and incubated for another hour with a goat anti-rabbit horseradish peroxidase–conjugated antibody 1:500 before development with ECL Detection (Amersham).

Balloon Injury and Immunodetection of PPAR in Rat and Human Vascular Lesions
Balloon-catheter injury was induced in male Sprague-Dawley rats.⁹ At 0, 2, 7, and 14 days after injury, aortas were removed, cut into cross-sectional segments, and embedded in paraffin.

Surgical specimens of human coronary artery lesions embedded in paraffin were obtained as approved by the Human Investigational Review Board at UCLA. Lesions were classified on the basis of their histological composition and structure in accordance with the report from the American Heart Association Committee on Vascular Lesions.²⁶

Sections were preincubated with a blocking buffer (PBS containing 5% BSA) for 60 minutes at room temperature. After incubation with polyclonal rabbit anti-PPAR (rabbit polyclonal), smooth muscle -actin (mouse monoclonal), rat macrophage marker ED1 (mouse monoclonal, Serotec), or human macrophage marker CD68 (goat polyclonal, Santa Cruz) in PBS containing 1% BSA for 60 minutes, biotinylated antibodies (Zymed) for 30 minutes, and streptavidin-peroxidase for 20 minutes, peroxidase activity was detected with an AEC kit (Zymed). Slides were counterstained with Mayer’s acid hematoxylin for 3 minutes.

Statistics
ANOVAs were performed and differences between means were determined by Student-Newman-Keuls test.Values of P<0.05 were considered statistically significant. Data are expressed as mean±SEM.

	Acknowledgments

 
This study was supported by NIH grant HL-58328-03 to W.A.H., Deutsche Forschungsgemeinschaftgrant DFG GO 800/1-1 to S.G., and ADA support to R.E.L. We thank Janie Teran and Dolores Mendoza for their assistance in preparing the manuscript.

	Footnotes

 
The Methods section of this article can be found at http://www.circulationaha.org

Received February 26, 1999; revision received September 22, 1999; accepted October 8, 1999.

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