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(Circulation Research. 2002;91:210.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Intimal Smooth Muscle Cells as a Target for Peroxisome Proliferator-Activated Receptor- Ligand Therapy

David Bishop-Bailey, Timothy Hla, Timothy D. Warner

From the Department of Cardiac, Vascular, and Inflammation Research (D.B.-B., T.D.W.), William Harvey Research Institute, Barts and the London, Queen Mary University of London, London, UK; and the Center for Vascular Biology, Department of Physiology (T.H.), University of Connecticut Health Center, Farmington, Conn.

Correspondence to David Bishop-Bailey, Dept of Cardiac, Vascular, and Inflammation Research, William Harvey Research Institute, Barts and the London, Queen Mary University of London, Charterhouse Square, London, UK EC1 M 6BQ. E-mail d.bishop-bailey{at}qmul.ac.uk

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activation of the nuclear receptor/transcription factor, peroxisome proliferator-activated receptor (PPAR), is a newly defined target for limiting vascular pathologies. PPAR is expressed in human and animal models of vascular disease, with particularly high levels being present in the cells of the neointimal microenvironment. In the present study, we show that intimal smooth muscle cells in vitro contain higher amounts of functional PPAR than medial smooth muscle cells. The PPAR ligand rosiglitazone more potently induced CD36 expression at low concentrations, and cell death by apoptosis at higher concentrations in intimal compared with medial smooth muscle cells. Intimal smooth muscle cells also contained high levels of cyclooxygenase-2 protein, and released a more diverse and larger amount of eicosanoids on arachidonic acid stimulation. Furthermore, when exogenous arachidonic acid was added, PPAR reporter gene activation was induced in a cyclooxygenase inhibitor–sensitive manner, an effect that correlated with an increase in CD36 expression. In summary, intimal smooth muscle cells contain functionally higher levels of PPAR, PPAR ligands have high- and low-potency targets in vascular smooth muscle cells, and cyclooxygenase can serve as a source of potential endogenous PPAR ligands. Intimal vascular smooth muscle cells therefore represent a potentially important target for the antiproliferative, and antiatherosclerotic actions of PPAR ligands.

Key Words: peroxisome proliferator-activated receptor-&ggr • cyclooxygenase-2 • intimal smooth muscle cells • atherosclerosis • restenosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptors (PPARs) are a family of 3 nuclear receptors: - (NR1C1), -ß/ (also referred to as NUC1 or NR1C2), and - (NR1C3), which heterodimerize with the retinoid X receptors (RXRs).¹ PPAR is found primarily in adipose tissue, where it plays a critical role in the differentiation of preadipocytes into adipocytes.^1,2 PPAR can be activated by a number of ligands,^3,4 including the antidiabetic thiazolidinediones (PPAR selective) ligands, and a number of eicosanoids, including 12-hydroxyeicosatetaenoic acid, 15-hydroxyeicosatetaenoic acid, 13-hydroxyoctadecadienoic acid, and the cyclooxygenase (COX) products, prostaglandin (PG) A₁, PGA₂, PGI₂, and PGD₂ and the dehydration product of PGD₂, 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂), oxidized low-density lipoprotein, and oxidized alkyl phospholipids.^3–5

PPAR is expressed in vascular smooth muscle cells (SMCs),^6–8 endothelial cells,^9,10 monocytes/macrophages,^11,12 and TH₁ lymphocytes.^13,14 Notably, PPAR is highly expressed in human¹⁵ and murine atherosclerotic lesions,¹⁶ as well as in the neointimal SMC layer of a rat arteries after balloon angioplasty damage.¹⁷ In contrast, the expression of PPAR in the medial SMC layer remains relatively low.¹⁷ PPAR ligands inhibit inflammatory cell responses^11–14; they inhibit the proliferation and migration of vascular smooth muscle cells in vitro,^7,8 atherosclerotic lesion development,¹⁸ and neointimal formation in vivo, ¹⁷ supporting a role for these receptors in the response to injury of the blood vessel wall.

Vascular smooth muscle cells exist in culture in different stable phenotypes. Most commonly utilized in culture is the "adult" medial spindle shaped SMC that grows typically with "hill and valley" morphology. However, probably of more relevance to the study of pathology and proliferative events in the blood vessel wall are the epithelial or "" SMC; cell types that have not been extensively studied in lipid signaling. These developmental SMC phenotypes can be isolated from neonatal rat aorta, but are re-expressed in the adult after vascular injury, where they form the neointima SMC layer.^19–25 These intimal SMC phenotypes differ from adult medial SMCs not only in morphology, but also in their ability to grow in plasma-derived serum, in their expression of PDGF-B, CYP1AI, elastin and osteopontin,^19,20,25 plasminogen activator,¹⁹ cellular retinal-binding protein-1, cytokeratin 8,²⁴ and in their relative low expression of 1 (I) collagen and PDGF -receptor.²⁶

As the in vivo evidence for the expression of PPAR in restenosis clearly links high expression to the newly formed neointima, we performed an in vitro analysis of intimal and medial SMC lines. The intimal SMCs "constitutively" express higher levels of PPAR, and inducible cyclooxygenase (COX-2),²⁷ and have exaggerated responses to PPAR ligands. These results indicate that the effects of PPAR ligands are selective toward intimal SMC compared with adult medial SMC responses, and correlate to the higher levels of protein expressed.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
15d-PGJ₂, arachidonic acid, COX-1, and COX-2 antisera were from Cayman Chemical Company; pEGFPN-1 was from Clontech; PPAR antisera for Western blots, ciglitazone, and WY14643 were from Biomol; ³H-PGE₂ and ¹⁴C-arachidonic acid were from Amersham Pharmacia Biotech; apoptosis ELISA, IL-1ß, D-luciferin, ATP, DTT, and tricine were from Roche; Effectene was from Qiagen; Trizol reagent, DMEM and anti-biotic/mycotics were from Life Technologies; Western blot luminescent detection reagents, PPAR-blocking peptide, antisera against PPAR, RXR, CD36, and HRP-conjugated anti-goat, anti-rabbit, and anti-mouse IgG were from Santa Cruz; FITC conjugated anti-goat IgG was from Cappell-ICN; pSV-ß-galactosidase, and RT-PCR enzymes were from Promega; Rosiglitazone, GW0072 and pACO.g.Luc were generous gifts from Dr Steven Smith (Glaxo SmithKline, Harlow, UK), Dr Timothy Willson (Glaxo SmithKline, Research Triangle, NC), and Dr Ruth Roberts (AstraZeneca, Macclesfield, UK), respectively; all other reagents were from Sigma-Aldrich.

Cell Culture
Polyclonal cell rat aortic vascular SMC lines WKY12-22, and WKY3m-22, WKY3m-26, and pac-1 were a gift from Dr David Han (University of Washington, Seattle). Primary rat aortic SMCs (RASMCs) were grown from explants or aorta derived from male Wistar rats as previously described,²⁸ and used between passage 3 to 5. Primary intimal SMCs derived from ballooned Wistar rats were a generous gift from Professor Giulio Gabbiani (University of Geneva, Switzerland). All cells were propagated in DMEM, containing 10% FBS, and supplemented with antibiotic/mycotic mix. All experiments were performed in serum-free medium, after cells had gone through an initially period of growth arrested for 24 house.

Western Blot
Western blot analysis using specific antibodies for COX-1 (1:1000) and COX-2 (1:1000) was performed as previously described.²⁹ Similar experiments were performed for the determination of PPAR (1:1000), RXR (1:200) and ß-actin. (1:1000)

Immunofluorescence
Immunofluorescent staining using specific antibodies for PPAR (1:50), or CD36 (1:100) was as previously described.^9,30 All images were taken after a 10-second exposure so that direct comparisons could be made to indicate changes in the intensity of staining.

RT-PCR
Total RNA was prepared using Trizol reagent according the manufacturer’s recommended protocol and converted to cDNA by standard methods. The rat CD36 primers (583 bp)³¹ were 5'-CAACAGCCT- TATCAAAAAGTC-3' and 5'-GCACACCATACGACGTACAG-3'. Rat glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was chosen as a control. G3PDH (452 bp)²⁸ primers were 5'-ACCACAGTC-CATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. Initial denaturing was done at 94°C for 3 minutes followed by 26 cycles (CD36 in intimal SMCs), 35 cycles (CD36 in medial SMCs), or 30 cycles (G3DPH both cell types) followed by 10 minutes at 72°C. For CD36, each cycle consisted of 30 seconds at 94°C, 30 seconds at 54°C, and 45 seconds at 72°C. For G3DPH, each cycle consisted of 35 seconds at 94°C, 35 seconds at 58°C, and 45 seconds at 72°C. PCR products were size fractionated with a 2% agarose gel and the bands visualized with ethidium bromide. Each PCR reaction resulted in a single band at the appropriate bp size. In parallel reactions where M-MLV RT was omitted, no bands were visible (data not shown).

Reporter Gene Activation
PPAR activation was measured as previously described^9,30 using the PPRE of the rat acyl CoA oxidase promoter, linked to drive expression of luciferase (pACOgLuc). Reporter gene activation was measured in a polyclonal selection of WKY12-22 cells stably expressing pACOgLuc (WKY12-22-ACO.Luc). WKY12-22 in 10-cm dishes were transfected with Effectene overnight using 1 µg pACOgLUC, and 0.5 µg pEGFPN-1, which contains a neomycin resistance cassette. Clones were then selected in 0.5 µg/dL G418 sulfate and pooled. Treatments were performed in 12-well plates. Cells were lysed with 200 µL of distilled H₂0 for 10 to 15 minutes and luciferase activity was measured in 100 µL of lysates, according to the manufacturer’s recommended protocol (Promega).

Viability, Proliferation, and Apoptosis Assays
Cell morphology was assessed by phase contrast using a Zeiss Axiovert TV100 microscope, and pictures taken using a SPOT II digital camera (Diagnostic Systems). Cell viability was measured after 48 hours of drug treatment using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.⁹ Apoptosis was assessed at 24 hours after drug treatment by either nuclear morphology using Hoechst 33258,⁹ or by commercially available ELISA that detects cytoplasmic histone-associated DNA complexes.

COX-2 Activity
The spectrum of prostanoid release from SMCs was initially characterized by thin-layer chromatography using ¹⁴C-labeled arachidonic acid.³² Individual PGE₂ and PGD₂ levels were measured in the conditioned culture supernatants by RIA^28,29 or by commercially available ELISA, respectively.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential Expression of PPAR in an Intimal Compared With Medial Vascular Smooth Muscle Cells
Adult medial cells WKY3m-22, WKY3m-26, pac-1, and primary RASMCs displayed a weak expression of PPAR throughout the cell (Figure 1). The only exception to this was in primary RASMCs where a small population of the cells (<10%) had PPAR in the nucleus, a finding that may indicate a greater heterogeneity of cell phenotypes in this population. The intimal SMC cell line WKY12-22 and primary rat intimal SMCs (iSMCs) contained a weak cytoplasmic expression, but displayed a strong nuclear expression of PPAR (Figure 1). This nuclear expression was also confirmed using confocal microscopy (data not shown) (see online Figure 1, which can be found in the online data supplement available at http://www.circresaha.org). In the absence of primary antibody (Figure 1), or when primary antibody was preabsorbed with blocking peptide (data not shown) (online Figure 1), no specific staining was observed.

View larger version (86K): [in this window] [in a new window]   Figure 1. Differential expression of PPAR in rat medial and intimal SMCs. Fluorescent micrographs (x1000) show the cellular expression of PPAR in (a) primary medial RASMC, (b) primary intimal SMC, (c) WKY3m-22 adult medial SMC, (d) WKY12-22 intimal SMC, (e) WKY3m-26 adult medial SMC, and (g) pac-1 adult medial SMC. f, Control lack of specific staining in WKY12-22 intimal SMC observed when primary antibody is omitted. White arrows indicate the strong nuclear staining of PPAR observed in intimal SMC types.

Because WKY12-22 and WKY3m-22 cells are intimal and medial phenotype cells from identical genetic backgrounds, and have the advantage over primary cultures of being stable cells lines, these SMCs were used as a model system to further confirm and compare these findings at the level of expression and activity.

Differential Expression of COX Enzymes, PPAR, and RXR in Intimal and Medial Vascular Smooth Muscle Cells
Expression of PPAR, its heterodimer binding partner, RXR, COX-1, and COX-2 were determined by Western blotting. Adult medial (WKY3m-22) SMCs contained COX-1, and low but detectable levels of COX-2, PPAR, and RXR. In contrast, intimal SMCs (WKY12-22), contained no detectable COX-1, but significantly higher levels of COX-2, PPAR, and RXR (Figure 2A). Both cells contained similar levels of ß-actin. Consistent with these findings, Northern blot analysis revealed that mRNA for PPAR is higher in WKY12-22 compared with WKY3m-22 SMC (data not shown) (online Figure 1). These results indicate that the WKY12-22 intimal SMC has higher levels of PPAR and COX-2, than the comparator medial SMCs. These findings are strikingly similar to the expression patterns of COX-2³³ and PPAR¹⁷ in the neointima formed in vivo after balloon angioplasty.

View larger version (27K): [in this window] [in a new window]   Figure 2. Differential expression of PPAR and COX enzymes in WKY3m-22 and WKY12-22 SMC. A, Expression of PPAR, its binding partner RXR, COX-1, COX-2, and ß-actin were measured by Western blot analysis in samples of equal protein from medial (M: WKY3m-22) and intimal (I: WKY12-22) SMCs. Data represent n=3 experiments. B, Thin-layer chromatography radiograph showing the capacity and spectrum of prostanoids [AA indicates arachidonic acid; HETEs; PGD2, PGE2, PGF2, and 6-k (6-keto)-PGF1 (the hydrolysis product of PGI2)], released by medial (M: WKY3m-22) and intimal SMC (I: WKY12-22) in response to 14C-arachidonic acid (15 µmol/L; 30 minutes).

Prostaglandin Release From Vascular Smooth Muscle Cells
We assessed the capacity of intimal and adult medial SMCs in culture to synthesize and release PGs from ¹⁴C labeled arachidonic acid using thin layer chromatography. Intimal SMCs released higher amounts of a broad range of PGs than adult medial SMC (Figure 2B), results that were confirmed by ELISA for PGD₂ (WKY3-m-22, 4±1 pg/dL per µg; WKY12-22, 12±3 pg/dL per µg protein; n=9; P<0.05) and radioimmunoassay for PGE₂ (WKY3m-22, 4±2 pg/dL per µg; WKY12-22, 96±9 pg/dL per µg protein; P<0.05). Similarly, basal and arachidonic acid–stimulated PGE₂ release was far greater in primary iSMCs than RASMCs (data not shown) (online Figure 2).

PPAR Ligands More Potently Induce Intimal SMC Responses
The effects of PPAR ligands were tested on the expression of CD36 and viability of intimal and adult medial SMC lines in culture. CD36 is a bona fide target for PPAR, as ligands are unable to induce CD36 expression in PPAR^-/- knockout cells.^34,35 In contrast, PPAR ligands induce growth arrest³⁸ and/or apoptosis⁹ in vascular cells, although the molecular targets linking PPAR to these growth inhibitory pathways in vascular cells are ill-defined.

Rosiglitazone induced concentration-dependent expression of CD36 protein (Figure 3A) and mRNA (Figure 3B) more potently in intimal (WKY12-22) than medial (WKY3m-22) SMCs. Concentrations of rosiglitazone above 1 µmol/L had no further significant effect on the induction of CD36 (data not shown). Similarly, rosiglitazone (Figure 4A), 15d-PGJ₂, and ciglitazone (data not shown) (online Figure 3) induced concentration-dependent cell death more potently in intimal (WKY12-22) than medial (WKY3m-22) SMCs. To test that supra-pharmacological concentrations of rosiglitazone induced cell death via PPAR, we used the partial agonist GW0072. Identical to the previously reported agonist and antagonist concentrations,³⁶ GW0072 induced concentration-dependent cell death at agonist levels (>1 µmol/L; Figure 4B), but completely inhibited rosiglitazone induced cell death at antagonist concentrations (<1 µmol/L; Figure 4B). The PPAR ligand induced cell death was via apoptosis, indicated by cytoplasmic rounding, nuclear condensation (data not shown), and an increase in the presence of cytoplasmic histone-associated DNA fragments, an early marker of cellular apoptosis. After 48 hours treatment, in intimal (WKY12-22) SMC rosiglitazone (100 µmol/L) induced a 5.30±0.03-fold (n=3), ciglitazone (10 µmol/L) a 3.34±0.78-fold (n=6), and 15d-PGJ₂ (3 µmol/L) a 7.15±4.65-fold (n=6) enrichment of cytoplasmic histone-associated DNA fragments relative to control.

View larger version (29K): [in this window] [in a new window]   Figure 3. Low concentrations of the PPAR ligand rosiglitazone more potently induce CD36 expression in intimal than medial SMCs. A, Fluorescent micrographs (x1000) showing the rosiglitazone (Rosi; 0.3 and 1 µmol/L) induced cellular expression of CD36 in intimal (WKY12-22) and medial (WKY3m-22) SMCs for 48 hours. 2oAb panels show the antibody control when experiments were performed as per protocol with the exception that the primary antibody was omitted. Weak cellular staining for CD36 is observed in untreated cells. Multiple pictures were taken for each treatment, and data shown are representative of n=8 from 3 experimental days. B, Semiquantitative RT-PCR showing the relative ratio of CD36 mRNA to constitutive G3DPH mRNA in intimal (filled bars, WKY12-22) and medial (open bars, WKY3m-22) SMCs treated with rosiglitazone (Rosi; 0.1, 0.3 and 1 µmol/L) for 6 hours. RT-PCR was performed as described in Materials and Methods section. Bands were analyzed using UTHSCSA Image Tool v.3 (n=3). *P<0.05 between control and rosiglitazone treated cells by 1-sample t test.

View larger version (21K): [in this window] [in a new window]   Figure 4. High concentrations of the PPAR ligand rosiglitazone more potently induce cell death in intimal than medial SMCs. A, Change in cell viability of intimal (filled squares; WKY12-22) and medial (open squares; WKY3m-22) SMC cell lines treated with rosiglitazone (0 to 100 µmol/L). B, Change in cell viability in intimal SMC (WKY12-22) treated with the partial PPAR agonist GW0072 alone (filled squares; 0.1 to 10 µmol/L) or in the presence of 30 µmol/L (filled circles) or 100 µmol/L (filled diamonds) rosiglitazone (Rosi). Cell viability was measured by the MTT assay after 48 hours, and expressed as a percent of control culture conditions. Data represent the mean±SEM of n=9 to 21 from 7 separate experiments.

In contrast to effects on CD36 expression where rosiglitazone acts at nmol/L, rosiglitazone induces apoptosis at µmol/L supra-pharmacological concentrations. These results show that the difference in protein levels of PPAR is clearly associated with potency of the ligands to both express CD36 and induce apoptosis.

Cyclooxygenase Mediates PPAR Activation in Intimal Smooth Muscle Cells
Using WKY12-22 cells stably expressing a PPAR reporter gene, we assessed whether activation of COX resulted in a subsequent activation of PPARs. Arachidonic acid (0 to 10 µmol/L) gave a significant increase in PPAR (Figure 5A). This concentration of arachidonic acid had no effect on cell morphology or viability (data not shown). When COX activity was blocked by piroxicam (10 µmol/L), an NSAID that does not directly interact with PPARs, ³⁷ arachidonic acid was unable to induce PPAR responses, indicating a COX product(s) as the PPAR activator. Moreover, arachidonic acid at concentrations that activated the PPRE (0 to 10 µmol/L) induced CD36 (Figure 5B). Expression of CD36 alone, or induced by rosiglitazone or arachidonic acid was inhibited by GW0072 at an antagonist concentration (Figure 6). Furthermore, GW0072 was also able to inhibit the PPAR reporter gene activity (given as relative light units/mg protein) induced by rosiglitazone or arachidonic acid: Control 6±2; 30 µmol/L rosiglitazone 41±16 (P<0.05 control versus treatment); 3 µmol/L arachidonic acid 35±10 (P<0.05 control versus treatment); 1 µmol/L GW0072 7±2; rosiglitazone+GW0072 9±4 (P<0.05 GW0072 versus treatment); and arachidonic acid+GW0072 12±2 (P<0.05 GW0072 versus treatment). Data were analyzed using ANOVA followed by Bonferroni post test.

View larger version (53K): [in this window] [in a new window]   Figure 5. Arachidonic acid induces PPAR responses in intimal SMCs. A, Induction of PPAR reporter gene activation in WKY12-22-ACO-Luc cells by arachidonic acid (0 to 10 µmol/L; AA), in the absence (filled bars) or presence (unfilled bars) of piroxicam (10 µmol/L). WKY12-22-ACO-Luc is a polyclonal intimal SMC cell line that stably expresses the rat acyl CoA oxidase PPRE linked to drive luciferase expression (see Materials and Methods). PPAR activation is measured by the increase in luciferase activity (relative light units x10-3) in cell lysates after 48 hours incubation. Data represent the mean±SEM of n=6 from 3 separate experiments. *P<0.05 control vs treated cells; P<0.05 piroxicam vs its respective treatment group as measured by 1-way ANOVA followed by Bonferroni post test. B, Fluorescent micrographs (x1000) showing the arachidonic acid (AA, 0 to 10 µmol/L)-induced cellular expression of CD36 in intimal (WKY12-22) SMCs treated for 48 hours. 2oAb panels show the antibody control when experiments were performed as per protocol with the exception that the primary antibody was omitted. Multiple pictures were taken for each treatment, and data shown are representative of n=6 from 3 experimental days.

View larger version (44K): [in this window] [in a new window]   Figure 6. Inhibition of rosiglitazone- and arachidonic acid–induced CD36 expression by antagonist concentrations of the PPAR partial antagonist GW0072. A, Fluorescent micrographs (x400) showing the inhibition by GW0072 (GW, 1 µmol/L) of the cellular expression of CD36 in medial (WKY3m-22) SMCs (Control), treated with rosiglitazone (Rosi, 1 µmol/L) or arachidonic acid (AA, 10 µmol/L) for 48 hours. Multiple pictures were taken for each treatment, and data shown are representative of n=3. Similar findings were obtained from intimal (WKY12-22) SMC. B, Semiquantitative RT-PCR showing the relative ratio of CD36 mRNA to constitutive G3DPH mRNA in intimal (WKY12-22) SMCs (Control) treated with either GW0072 (GW, 1 µmol/L), rosiglitazone (Rosi, 1 µmol/L), or rosiglitazone and GW0072 (+GW) for 6 hours. RT-PCR was performed as described in methods section. Bands were analyzed using UTHSCSA Image Tool v.3 n=3. *P<0.05 GW0072 vs control groups by 1-sample t test (Control-GW0072), or by unpaired t test (rosiglitazone-rosiglitazone+GW0072). Similar results were found in medial (WKY3m-22) SMCs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we show that intimal SMCs contain significantly more active PPAR than corresponding medial phenotype SMCs. These in vitro findings directly correlate with the pattern of both intimal SMC and PPAR expression found in human and animal vascular lesions. Clearly, this suggests that drugs selective for PPAR could be targeted to these cells with the aim of reducing lesion development.

In atherosclerosis, there is increased PPAR expression throughout intimal and neointimal SMC layers.^8,15,16 In rat models of restenosis after balloon injury, intimal thickening is due to SMC remodeling, with few inflammatory cell components. PPAR in these studies is expressed exclusively in this neointimal vascular smooth muscle layer.¹⁷ Because the neointimal lesion here is considered primarily due to the proliferation of the intimal vascular SMC,^19–26 we suggest that the increase in PPAR expression seen in these lesions is at least due in part to the re-expression and proliferation of these cells. These findings are not without precedent as a number of in vitro markers for intimal SMC, such as plasminogen activator,²¹ cellular retinal binding protein-1, cytokeratin 8,²⁴ and osteopontin,^19,20,25 are also found highly expressed in the neointima in vivo. With respect to PPAR expression and activation it would, therefore, also seem that WKY12-22 SMCs and primary iSMCs are representative of cells present in the intimal environment in vivo.

Although, a number of stable characteristics of intimal smooth muscle cells in vivo are maintained in vitro, mediators such as M-CSF, GM-CSF, oxidized LDL, or PPAR ligands themselves can also increase the expression of PPAR.¹⁵ The effect of these mediators cannot, therefore, be discounted when one considers the levels of in vivo expression of the receptor. However, at least in terms of restenosis after angioplasty, where the intimal smooth muscle seem almost exclusively to form the neointima, it is clear that if an increase in expression of PPAR does occur due to these stimuli, it is also selectively occurring in intimal SMCs.

A number of studies have investigated the effects of PPAR ligands on adult medial SMC in vitro. These have indicated that PPAR ligands can cause inhibition of adult SMC migration and proliferation^7,8 by inducing cell cycle arrest at the G1-S interface.³⁸ PPAR ligands also inhibit angiotensin II type 1 receptor expression,³⁹ upregulate CD36,⁴⁰ and type II secretory PLA₂,⁴¹ and decrease MMP-9 expression.⁷ Although ligands have many effects in vitro, the overall effect of PPAR ligand therapy in animal models of vascular damage and inflammation does appear to lead to beneficial responses.¹⁸ Our study therefore used a simple model system to investigate the pharmacological differences between the different intimal and medial SMC phenotypes. PPAR ligands were more effective both at inducing CD36 expression and causing apoptosis in intimal compared with medial SMCs.

All experiments were performed in the absence of serum, or other mitogens used to activate SMCs, and under these conditions SMCs directly undergo apoptosis when treated with high concentrations of PPAR ligands. In SMCs activated by PGDF and insulin, PPAR ligands induce cell cycle arrest with little evidence of apoptosis.³⁸ In our experiments, SMCs were already in a quiescent state and rapidly under went apoptosis when treated with PPAR ligands, although cyclin-dependent kinase inhibitor p21 induction may precede this apoptosis (unpublished observation, 2000). Although the exact mechanisms for the beneficial effects of PPAR ligands’ ability to inhibit atherosclerosis and restenosis in vivo is not known, our results suggest that the high levels of PPAR found in vascular lesion sites could provide a pharmacological differences that could be exploited by selective ligands.

Interestingly, rosiglitazone was a very weak inducer of cell death. These results are in contrast to its effect on CD36 expression, where rosiglitazone is 100 to 1000 times more potent. The use of cells that are genetically PPAR deficient,^34,35 clearly demonstrate that PPAR ligands can have antiinflammatory and antiproliferative properties independent of PPAR. The use of high concentrations of thiazolidinediones in this respect has therefore been controversial. However, our data support the contention that the actions of supra-pharmacological concentrations of rosiglitazone on apoptosis are still PPAR dependent, and indicate that there are concentration-dependent targets for rosiglitazone within SMCs. Theoretically, this is possible either by specificity of the activated PPAR for different target PPREs,^42–45 or by the concentration-dependent recruitment of different coactivators.^46,47 In this respect, each PPAR ligand to a varying degree may have selective PPAR modulator (SPRM) activity on different targets, an acronym used to describe N-(9-fluorenylmethyloxycarbonyl)-L-Leu, a PPAR agonist that acts as an insulin sensitizer at a greater potency than it causes adipogenesis.⁴⁷

Like PPAR, COX-2 was expressed in higher amounts in intimal SMCs than in medial SMCs, whereas the opposite expression pattern was observed for COX-1. This COX was active, because the WKY12-22 cells released greater amounts of PGs under basal conditions, or when directly activated by ¹⁴C-arachidonic acid. Furthermore, COX-2–like PPAR is highly expressed in human atherosclerotic lesions,^48,49 animal models of vascular damage, ⁵⁰ and particularly at high levels in the neointima after balloon angioplasty in the rat.³³ Vascular SMCs release large amounts of PGE₂^28,29,51 from COX-2, and in the present study, we show that in vitro they are capable of releasing a wide variety of other arachidonic acid products including PGD₂. COX products can both activate and inhibit PPAR. The dehydration products of PGD and PGE series prostaglandins, the PGJ, and PGA series cyclopentanone prostaglandins, respectively, can serve as PPAR ligands,^3,4 whereas PGF₂ can inhibit PPAR activity via a MAP kinase–induced phosphorylation, mediated by activation of its cell surface FP receptor.⁵² When intimal cells were treated with the COX substrate arachidonic acid, PPAR reporter gene was activated and CD36 was induced. A COX product does appear to be serving as a PPAR ligand, as the arachidonic acid reporter gene activation was inhibited by the COX-inhibitor piroxicam, a NSAID with no reported PPAR activity.³⁷ Similar to rosiglitazone, the arachidonic acid induced reporter gene activation and CD36 expression was inhibited by GW0072 at antagonist concentrations.

In summary, developmental/intimal SMCs contains more active PPAR and COX-2 than corresponding adult/medial SMC lines. Consequently, PPAR ligands, which may include endogenous COX products, are more potent at producing functional PPAR-mediated responses in intimal SMCs. Because there is increasing evidence that intimal SMCs play an important role in the pathology of vascular diseases, these intimal SMCs may represent a therapeutic target for PPAR ligand therapy, and may provide a particularly good in vitro model system to study the ways in which PPAR ligands exert antiatherosclerotic and antirestenotic effects.

	Acknowledgments

 
Dr Bishop-Bailey is a British Heart Foundation Intermediate Fellow (FS/99047).

	Footnotes

 
Dr Bishop-Bailey received an honorarium from Takeda for a talk about PPAR ligands and their effect on intimal proliferation and endothelial function.

Received January 24, 2002; revision received June 25, 2002; accepted June 26, 2002.

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