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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:52.)
© 2003 American Heart Association, Inc.

Vascular Biology

Peroxisome Proliferator-Activated Receptor Ligands Increase Release of Nitric Oxide From Endothelial Cells

David S. Calnek; Louis Mazzella; Susanne Roser; Jesse Roman; C. Michael Hart

From the Department of Medicine, Veterans Affairs and Emory University, Medical Centers, Decatur, Ga.

Correspondence to David S. Calnek, PhD, Atlanta VA Medical Center (151P), 1670 Clairmont Rd, Decatur, GA 30033. E-mail dcalnek{at}hotmail.com

	Abstract

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Objective— Peroxisome proliferator-activated receptor (PPAR) ligands reduce lesion formation in animal models of atherosclerosis by mechanisms that have not been defined completely. We hypothesized that PPAR ligands stimulate endothelial-derived nitric oxide release (·NO) to protect the vascular wall.

Methods and Results— The PPAR ligands, 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) or ciglitazone, stimulated a PPAR response element-luciferase reporter construct in transfected porcine pulmonary artery endothelial cells (PAECs), demonstrating that PPAR was transcriptionally functional. Treatment with 15d-PGJ₂ or ciglitazone significantly increased release of ·NO from PAECs or human aortic endothelial cells and augmented calcium ionophore–induced ·NO release from human umbilical vein endothelial cells measured by chemiluminescence analysis of culture media. Increases in ·NO release caused by treatment with 15d-PGJ₂ occurred at 24 hours, but not after 1 to 16 hours, and were abrogated by treatment with the transcriptional inhibitor -amanitin. Overexpression of PPAR or treatment with 9-cis retinoic acid also enhanced PAEC ·NO release. Neither 15d-PGJ₂ nor ciglitazone altered eNOS mRNA, whereas 15d-PGJ₂, but not ciglitazone, decreased eNOS protein.

Conclusions— Taken together, these findings demonstrate that PPAR ligands stimulate ·NO release from endothelial cells derived from multiple vascular sites, through a transcriptional mechanism unrelated to eNOS expression.

Key Words: peroxisome proliferator-activated receptor • endothelium • nitric oxide • nitric oxide synthase • thiazolidinedione

	Introduction

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The production of nitric oxide (·NO) by vascular endothelial cells is critical for maintenance of normal vascular physiology.¹ In endothelial cells (ECs), the type III endothelial nitric oxide synthase (eNOS) produces ·NO from the amino acid L-arginine. Our preliminary observations,² as well as reports by others,^3–5 indicate that exogenous fatty acids alter EC ·NO production. The molecular mechanism contributing to fatty acid–induced alterations in EC ·NO production remain unexplored. One potential mechanism for fatty acid–induced alterations in gene expression is the activation of peroxisome proliferator-activated receptors (PPARs). Originally described in 1990, PPARs belong to the nuclear hormone receptor superfamily of ligand-activated transcription factors including steroid, thyroid, and retinoid hormone receptors.⁶ Structurally diverse ligands including long-chain fatty acids, eicosanoids, thiazolidinediones, and fibrates activate PPARs, which form obligate heterodimers with the 9-cis retinoic acid receptor, RXR.⁷ On ligand binding, PPARs become transcriptionally active at PPAR response elements (PPRE) and alter the expression of target genes.

PPAR is expressed in vascular endothelial cells^8–11 and smooth muscle cells.¹² The expression of PPARs in vascular wall cells suggests their potential role in vascular disease.^8–10 Some in vitro studies suggest potential atherogenic effects of PPAR activation,^8,13–15 whereas other studies associate PPAR with potential vascular protective effects.^16–21 Importantly, two independent in vivo studies using the LDL receptor knockout mouse demonstrated that PPAR activators reduced development of hypercholesterolemia-induced atherosclerotic lesion formation.^22,23 These results demonstrate that PPAR activation has the potential to exert antiatherogenic effects in animal models of atherosclerosis.

The insulin-sensitizing thiazolidenedione class of drugs, including troglitazone, ciglitazone, rosiglitazone, and pioglitazone, activates PPAR. These medications improve diabetic metabolic derangements associated with atherogenesis. The present study examines whether PPAR ligands also exert direct effects on vascular endothelial cells. Our results demonstrate that PPAR is expressed in ECs derived from porcine pulmonary artery and human umbilical vein, that these ECs respond to the PPAR ligands 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) and ciglitazone by increasing ·NO release, and that overexpression of PPAR stimulates EC ·NO release. This report describes for the first time a direct link between PPAR ligands and stimulation of endothelial ·NO release, thereby providing novel insights into direct vascular effects of PPAR ligands.

	Methods

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Cell Culture
Porcine pulmonary artery endothelial cells were isolated and maintained in culture, as we have previously reported.^24–27 Human umbilical vein endothelial cell (HUVEC) or human aortic EC (HAEC) monolayers were maintained in phenol red-free EGM (endothelial growth medium) according to protocols provided by the manufacturer (Clonetics). In all experiments, ECs were studied 2 to 4 days after becoming confluent. PAECs, HAECs, or HUVECs were treated with graded concentrations (0.01 to 10 µmol/L) of PPAR agonists, including 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) (Calbiochem) or ciglitazone (Biomol), or with the RXR ligand, 9-cis retinoic acid (Sigma Chemical Co), -amanitin (Sigma Chemical Co), or ethanol vehicle (0.1%, vol:vol) in maintenance medium (MEM with 4% FBS) for 24 hours unless indicated otherwise. Because 15d-PGJ₂ can exert PPAR-independent effects,^28,29 we used several ligands to strengthen our conclusions regarding PPAR-dependent effects.

PPAR and eNOS Expression
EC PPAR expression was determined by measuring PPAR protein levels in crude nuclear extracts (CNEs).³⁰ Protein concentrations were determined by the Bradford method (BioRad). CNE or whole-cell homogenates were subjected to SDS polyacrylamide gel electrophoresis (4% to 12% gradient gels) followed by electroblotting of proteins onto PVDF membranes. After appropriate blocking, the blots were probed with commercially available primary monoclonal or polyclonal antibodies to PPAR (Santa Cruz Biotechnology, Inc) or eNOS (Transduction Labs). Relative immunoreactive levels of PPAR or eNOS were determined using a laser densitometer (model GS-800, BioRad).

The effect of PPAR ligands on eNOS message expression was examined with a bioluminescent detection assay for RNA. RNA was extracted from PAEC using the reagent RNAzol B (Tel-Test Inc) followed by purification with serial chloroform, isopropanol, and ethanol extractions. The mRNA was reverse transcribed to cDNA using SuperScript II RT (GibcoBRL), a cDNA "Master Mix" containing dNTPs (GibcoBRL), random Hexamer oligonucleotides (Roche Molecular Biochemicals), and Rnasin, an RNase inhibitor (Promega). Amplification of the reverse-transcribed product was achieved using 5'-biotinylated (forward) primers. Primers to eNOS and the housekeeping gene, GAPDH, were synthesized by Sigma-Genosys based on sequences obtained from GeneBank (5' to 3'): NOS F-primer, CAC CTG ATC CCA GCT TGC; NOS R-primer, CGT CCA GCT CCA TGT TGC; NOS probe, GAG GAC GCG TCC AAA CAC; GAPDH F-primer, AAG GCT GGG GCT CAC TTG; GAPDH R-primer, CCA CAA CCTG ACA CGT TGG; GAPDH probe, GGT GGC AGT GAT GGC ATG. The biotinylated primer–polymerase chain reaction (PCR) products were captured on streptavidin-coated plates (Roche Molecular Biochemicals) and probed with Digoxigenin (Dig)-labeled probes. Anti-Dig antibody labeled with the bioluminescent molecule aequorin (AquaLite, SeaLite Sciences) was added, and calcium-induced luminescence was measured on a luminometer. Standard curves were performed to ensure that the amounts of cDNA being amplified had not reached a plateau in the amplification curve for any primer pair and number of cycles. All results were normalized to the expression of the housekeeping gene, GAPDH.

EC Transfection
ECs were transfected with pPPRE₃tk-luc,³¹ a reporter plasmid in which luciferase expression is induced by PPAR agonists (obtained as a generous gift from Dr Ronald Evans, Salk Institute). This procedure produced a transfection efficiency of 10%. Once confluent, transfected or mock-transfected ECs were treated with PPAR agonists. In separate studies to elicit overexpression of PPAR, pCMXPPAR³² (obtained as a kind gift from Dr Ronald Evans, Salk Institute) was transfected into ECs as described above. Western analysis was performed to determine the level of PPAR expression in PPAR- and mock-transfected ECs.

Chemiluminescence Analysis of EC ·NO Release
To measure NO production, aliquots of culture medium were collected from ECs, centrifuged, and subjected to chemiluminescence analysis of ·NO and its oxidation products, as we have previously reported.²⁴ Standard curves with NaNO₂ were performed daily.

Statistical Analysis
All experiments were analyzed with analysis of variance to determine the significance of treatment effects, followed by Student Newman-Keuls analysis to examine differences between individual treatment groups. The level of significance was taken as P<0.05.

	Results

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PPAR Activators Stimulate ·NO Release in EC
PPAR expression was not altered in EC by either 15d-PGJ₂ or ciglitazone treatment (data not shown). To determine if treatment with the PPAR ligands causes functional activation of PAEC PPAR, PAECs were transfected with pPPRE₃tkluc followed by treatment with 15d-PGJ₂ or ciglitazone for 24 hours. Luciferase activity was monitored and normalized to ß-gal activity of a cotransfected pCMV-ß-galactosidase expression vector. Treatment with 10 µmol/L 15d-PGJ₂ or ciglitazone caused 1.8-fold or 2.1-fold increases, respectively, in relative luciferase activity (Figure 1A). These results indicate that either 15d-PGJ₂ or ciglitazone increased endogenous PPAR-dependent transcriptional responses in PAECs. Treating PAECs for 24 hours with graded concentrations of '31'35d-PGJ₂ or the thiazolidenedione ciglitazone increased ·NO production compared with control PAEC (Figure 1B). Studies examining the time course of PPAR ligand–stimulated PAEC ·NO production demonstrated that 15d-PGJ₂ tended to increase PAEC ·NO release at 16 hours, but this stimulation achieved statistical significance only at 24 hours (Figure 1C). Because PPARs can directly modulate gene transcription, we examined the effects of -amanitin, a global RNA synthesis inhibitor, on ·NO release, in the absence or presence of 15d-PGJ₂. Treatment with -amanitin attenuated 15d-PGJ₂–stimulated ·NO release to levels comparable with control treatment (Figure 2), indicating that PPAR regulates ·NO release through changes in gene transcription.

View larger version (19K): [in this window] [in a new window]   Figure 1. ECs increase PPAR functional activity and enhance ·NO release in response to PPAR ligands. A, PAECs were cotransfected with pCMV-ßgal and a luciferase reporter gene vector under the control of 3 PPAR response elements. After 48 hours, PAECs were treated with graded concentrations of 15d-PGJ2 or ciglitazone for 24 hours. Each bar represents the mean luminescence (in arbitrary units) ±SEM (n=3). B, PAECs were treated for 24 hours with graded concentrations of 15d-PGJ2 (n=10) or ciglitazone (n=8), as indicated. ·NO release was measured with chemiluminescence analysis of culture media and normalized to PAEC protein content. Each bar represents mean ·NO release expressed as fold induction above control±SEM. C, PAECs were incubated for indicated times with 10 µmol/L 15d-PGJ2. ·NO release was normalized to PAEC protein content. Each bar represents mean ·NO release as fold induction above control±SEM (n=4) *P<0.05 compared with control cells treated with vehicle (0.1% ethanol).

View larger version (10K): [in this window] [in a new window]   Figure 2. -Amanitin attenuates 15d-PGJ2–stimulated PAEC ·NO release. PAECs were treated for 24 hours with 10 µmol/L 15d-PGJ2 (PGJ2) or 1 µg/mL -amanitin (AA) followed by 1 hour incubation in serum-free EBM. Media were analyzed for NO release by chemiluminescence and normalized to total cell protein. Each bar represents the mean ·NO release as fold induction above control±SEM (n=3). *P<0.05 compared with control PAECs treated with vehicle (0.1% ethanol). **P<0.05 compared with 15d-PGJ2–treated cells.

To determine if PPAR ligands exerted similar effects on ·NO release in human ECs, HAECs were treated with graded concentrations of 15d-PGJ₂ for 24 hours. As illustrated in Figure 3A, 10 µmol/L 15d-PGJ₂ stimulated HAEC ·NO release. To determine if PPAR ligands stimulated not only basal ·NO release, but also augmented ionophore-stimulated ·NO release, HUVECs were treated with graded concentrations of either 15d-PGJ₂ or ciglitazone for 24 hours followed by treatment with the calcium ionophore A23187 (5 µmol/L) for 30 minutes. A23187 alone stimulated ·NO release in HUVECs (mean ·NO release±SEM; control, 32.6±6.1; A23187, 57.7±9.4 pmol/min per mg protein; n=7). As illustrated in Figure 3B, treatment with either 15d-PGJ₂ or ciglitazone enhanced A23187-stimulated ·NO release. Because the RXR receptor forms obligate heterodimers with PPARs, 9-cis retinoic acid (9-cis RA), a retinoid that binds and activates the RXR receptor, was examined for its ability to modulate ·NO release in ECs. Treatment with 9-cis RA for 24 hours increased PAEC ·NO release (Figure 4) compared with the increases seen with similar concentrations of 15d-PGJ₂ (Figure 1B).

View larger version (17K): [in this window] [in a new window]   Figure 3. PPAR ligands stimulate human EC ·NO release. A, HAECs were treated for 24 hours with graded concentrations of 15d-PGJ2, and then ·NO release was measured for 1 hour as described in Methods (n=3). B, HUVECs were treated for 24 hours with graded concentrations of either 15d-PGJ2 or ciglitazone, followed by treatment for 0.5 hours with 5 µmol/L A23187. Media were then analyzed for NO release by chemiluminescence and normalized to total cell protein. ·NO release is presented as the mean fold induction above control±SEM (n=3). *P<0.05 compared with control ECs treated with vehicle (0.1% ethanol).

View larger version (14K): [in this window] [in a new window]   Figure 4. The RXR receptor agonist, 9-cis retinoic acid, increased basal PAEC ·NO release. After 24-hour exposure to 9-cis retinoic acid, PAECs were incubated for 1 hour in serum-free medium that was collected for analysis of ·NO release by chemiluminescence, as described in Methods. Each bar represents the mean ·NO release (as fold induction compared with control cells treated with vehicle±SEM [n=3]). *P<0.05 compared with control.

Effect of PPAR Ligands on eNOS Expression
To determine if PPAR regulates NO release through alterations in eNOS expression, PAECs were treated for 24 hours with 15d-PGJ₂ or ciglitazone, followed by reverse transcriptase–PCR and Western blot analysis for measuring eNOS message and protein levels, respectively. Figure 5A illustrates that neither 15d-PGJ₂ nor ciglitazone significantly altered eNOS mRNA relative to GAPDH mRNA, as measured in normalized light units. However, Western blot analysis demonstrated that eNOS protein levels in PAEC and HUVEC were decreased by treatment with 10 µmol/L 15d-PGJ₂ for 24 hours, whereas ciglitazone had no significant effect on eNOS protein expression (Figures 5B and 5C). The inducible type 2 NOS isoform (iNOS) was not detected by Western blot analysis in lysates derived from PAECs or HUVECs treated with either 15d-PGJ₂ or ciglitazone (data not shown). These results indicate that PPAR-stimulated ·NO release is not attributable to increased eNOS or iNOS expression.

View larger version (15K): [in this window] [in a new window]   Figure 5. PAEC and HUVEC eNOS expression after treatment with PPAR ligands for 24 hours. PAECs and HUVECs were treated for 24 hours with the PPAR ligands 15d-PGJ2 or ciglitazone (10 µmol/L). Cell extracts were then analyzed for eNOS expression, either by reverse transcriptase–PCR (A, PAEC only) or Western blot analysis (B, PAECs and HUVECs). A, All reverse transcriptase–PCR values represent mean light units normalized to GAPDH expression levels±SEM (n=2). B, Each bar represents mean±SEM relative eNOS densitometric units compared with control cells treated with vehicle (0.1% ethanol) only (PAEC, n=10; HUVEC, n=12). *P<0.05 compared with control.

Effect of PPAR Overexpression on Basal PAEC ·NO Release
To additionally confirm the role of PPAR in regulating ·NO release, PAECs were transfected with the expression vector pCMXPPAR. Western blot analysis of transfected cells confirmed that PPAR expression was increased 3.5-fold over mock-transfected cells (Figures 6A and 6B). PPAR overexpression in vascular endothelial cells significantly increased ·NO release with or without exogenous PPAR ligands (10 µmol/L for 24 hours) compared with untreated mock-transfected cells (Figure 6C). These results additionally demonstrate that PPAR plays a role in the enhancement of endothelial ·NO release.

View larger version (24K): [in this window] [in a new window]   Figure 6. Overexpression of PPAR increased PAEC ·NO release. After transfection with pCMXPPAR, PPAR expression and ·NO production were examined. A, Western blotting for PPAR was performed on in vitro translated PPAR (IVT), Jukat cell nuclear extracts, PAEC lysates of mock transfection, and PAEC lysates collected 48 hours after transfection. The reason that the molecular mass of PPAR derived from pCMXPPAR (IVT or transfected) is slightly larger than endogenously expressed Jurkat or PAEC is unknown. Densitometric analysis of PPAR expression in mock- or pCMXPPAR-transfected PAECs is represented in B. C, After treatment with 10 µmol/L 15d-PGJ2 (PGJ2) or ciglitazone (Cig) for 24 hours, cell culture media from mock- or pCMXPPAR-transfected PAECs was analyzed for ·NO release with chemiluminescence, as described in Methods. Each bar represents the mean densitometric intensity (B) ±SEM (n=4) or mean ·NO release in pmol/min per mg protein (C) ±SEM (n=3). * P<0.05 compared with mock control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The expression of PPAR receptors in vascular endothelial cells provides a potential mechanism by which circulating signals from structurally diverse ligands might be integrated at the level of the vascular wall. In vascular endothelium, PPAR functions to increase Cu/Zn superoxide dismutase³³ and plasminogen activator inhibitor-1 expression⁸ and inhibit cytokine-induced monocyte chemotactic protein-1 production,³⁴ leukocyte-endothelial interactions,¹⁰ angiogenesis,¹¹ and thrombin-induced endothelin-1 production.⁹ Taken together, these reports suggest that PPAR plays an important role in endothelial biology and the pathogenesis of vascular disease.

This report expands the recognized effects of PPAR ligands on the vascular endothelium to include enhanced ·NO release and describes for the first time the expression of PPAR in ECs derived from the pulmonary artery. However, similar stimulation of ·NO release by PPAR ligands in PAEC, HAEC, and HUVEC suggests that these effects may be generalized to all macrovascular ECs, regardless of species or vascular bed from which they are derived. The role of PPAR in vascular endothelium and the downstream events regulated by ligands of these receptors continues to be clarified. Because ·NO participates in the regulation of vessel tone,³⁵ platelet adhesion and aggregation,³⁶ adhesion molecule expression,³⁷ endothelin-1 secretion,³⁸ and smooth muscle cell proliferation,^39,40 PPAR-induced stimulation of EC ·NO release provides a novel, and potentially unifying, mechanism for the direct vascular protective effects of PPAR ligands, including thiazolidinedione medications. The mechanism by which PPAR ligands lead to increased EC ·NO release remains to be defined. In contrast to our findings that PPAR agonists enhance basal and ionophore-stimulated endothelial ·NO release, Ikeda et al⁴¹ demonstrated that PPAR ligands inhibited IL-1ß–stimulated ·NO levels in vascular smooth muscle cells. This effect was attributed to decreased iNOS expression meditated by suppression of nuclear factor (NF)-B activity. However, in our study, iNOS was not detected in ECs treated with PPAR activators (data not shown), suggesting that other iNOS-independent mechanisms account for the ability of PPAR to modulate endothelial ·NO release.

Several lines of evidence suggest that the ability of PPAR ligands to stimulate ·NO release does not represent simple nonspecific EC activation. For example, the PPAR ligands used in this study, 15d-PGJ₂ and ciglitazone, caused comparable increases in PAEC ·NO release but represent structurally diverse molecules that stimulated PPAR functional activity to a comparable degree (Figure 1A) without altering PPAR expression (data not shown). 15d-PGJ₂ can exert PPAR-independent effects, including inhibition of IB kinase and interruption of NF-B signaling.^28,29 Such PPAR-independent effects are suggested by the ability of 0.1- to 1.0-µmol/L concentrations of 15d-PGJ₂ to significantly increase ·NO release without stimulating PPRE-mediated reporter activity (Figures 1A and 1B). However, the stimulating effects of 15d-PGJ₂ on endothelial ·NO release occurred only after 16 hours and were inhibited by the transcriptional inhibitor -amanitin. These findings indicate that structurally unrelated PPAR ligands stimulate EC ·NO release through transcriptional activation and, likely, time-dependent translational activation. Furthermore, 9-cis retinoic acid stimulated PAEC ·NO release (Figure 4). The ability of RXR ligands to stimulate PPAR-mediated effects⁴² suggests that 9-cis retinoic acid is acting, at least in part, through a PPAR signaling pathway. Finally, EC ·NO release was stimulated not only by PPAR ligands but also by overexpression of PPAR. Control cells overexpressing PPAR released greater levels of ·NO compared with their mock-transfected counterparts, suggesting that increased ·NO release was attributable to endogenous ligand. The inability of PPAR ligands to stimulate additional increases in ·NO release from ECs overexpressing PPAR (Figure 6C) remains unexplained but suggests that the capacity to increase ·NO release by this mechanism is not unlimited. Taken together, these results provide strong evidence that PPAR ligands stimulate EC ·NO release.

Localized within the eNOS promoter are a variety of cis-acting elements that interact with specific transcription factors.^43,44 Analysis of the eNOS promoter sequence did not reveal the presence of any discernible PPAR response elements. Because neither 15d-PGJ₂ nor ciglitazone treatment increased eNOS mRNA or protein levels, it seems likely that PPAR ligands regulate the expression of another target gene that promotes increased EC ·NO release, particularly because 15d-PGJ₂ increased ·NO release but decreased eNOS protein expression. The mechanisms for these effects of 15d-PGJ₂ remain undefined but are reminiscent of the reported ability of inflammatory cytokines to simultaneously decrease eNOS expression in HUVECs and stimulate ·NO production through enhanced tetrahydrobiopterin production.⁴⁵ Therefore, it seems likely that PPAR ligands regulate the expression of other gene products, which, in turn, increase ·NO bioavailability. For example, PPAR ligands could potentially regulate the expression of other proteins such as caveolin⁴⁶ or hsp90⁴⁷ that complex with eNOS to regulate its activity. Alternatively, Inoue et al³³ demonstrated that PPAR ligands increased EC Cu/Zn superoxide dismutase mRNA levels and decreased PMA-stimulated p47phox expression. Thus, PPAR ligands could also potentially decrease EC superoxide levels to enhance ·NO bioavailability. Identification of the gene target involved in PPAR-mediated regulation of EC ·NO bioavailability constitutes an active area of investigation in our laboratory.

The ultimate impact of PPAR ligands on the pathogenesis of vascular disease will likely depend on the cumulative influence of their systemic effects on lipid metabolism and inflammation as well as their local and direct effects on the vascular wall. Animal studies have provided valuable insights into the overall vascular wall effects of PPAR activation. The thiazolidinedione rosiglitazone decreased arterial lesions in LDL-receptor knockout mice³⁷ and improved impaired endothelial-dependent vasodilation in fatty Zucker rats,³⁶ suggesting overall vascular protective effects in these animal models. Our findings suggest that PPAR-stimulated endothelial ·NO release may contribute to these vascular protective effects. In addition, our results demonstrate that PPAR expression and functional activation in ECs derived from the pulmonary circulation could indicate novel potential targets for the pharmacologic management of pulmonary hypertension. However, the potential application of PPAR ligands to the prevention or treatment of vascular disease will require additional studies to better characterize the molecular targets regulated by PPAR as well as their impact on clinical outcomes.

	Acknowledgments

 
This work was supported in part by grants from the Veterans Affairs Research Service (to Dr Hart), the National Institutes of Health (DK 61274, to Dr Hart), and the American Heart Association Midwest Affiliate with cosponsorship with the Southeast Affiliate (0030387Z, to Dr Calnek). The authors gratefully acknowledge the expert technical assistance of Dean Kleinhenz.

Received August 7, 2002; accepted September 18, 2002.

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