Peroxisome Proliferator-Activated Receptor γ Ligands Increase Release of Nitric Oxide From Endothelial Cells
Objective— Peroxisome proliferator-activated receptor γ (PPARγ) ligands reduce lesion formation in animal models of atherosclerosis by mechanisms that have not been defined completely. We hypothesized that PPARγ ligands stimulate endothelial-derived nitric oxide release (·NO) to protect the vascular wall.
Methods and Results— The PPARγ ligands, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, stimulated a PPAR response element-luciferase reporter construct in transfected porcine pulmonary artery endothelial cells (PAECs), demonstrating that PPARγ 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 α-amanitin. Overexpression of PPARγ 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 PPARγ ligands stimulate ·NO release from endothelial cells derived from multiple vascular sites, through a transcriptional mechanism unrelated to eNOS expression.
- peroxisome proliferator-activated receptor γ
- nitric oxide
- nitric oxide synthase
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.
PPARγ is expressed in vascular endothelial cells8–11⇓⇓⇓ 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 PPARγ activation,8,13–15⇓⇓⇓ whereas other studies associate PPARγ with potential vascular protective effects.16–21⇓⇓⇓⇓⇓ Importantly, two independent in vivo studies using the LDL receptor knockout mouse demonstrated that PPARγ activators reduced development of hypercholesterolemia-induced atherosclerotic lesion formation.22,23⇓ These results demonstrate that PPARγ 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 PPARγ. These medications improve diabetic metabolic derangements associated with atherogenesis. The present study examines whether PPARγ ligands also exert direct effects on vascular endothelial cells. Our results demonstrate that PPARγ is expressed in ECs derived from porcine pulmonary artery and human umbilical vein, that these ECs respond to the PPARγ ligands 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone by increasing ·NO release, and that overexpression of PPARγ stimulates EC ·NO release. This report describes for the first time a direct link between PPARγ ligands and stimulation of endothelial ·NO release, thereby providing novel insights into direct vascular effects of PPARγ ligands.
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 PPARγ agonists, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) (Calbiochem) or ciglitazone (Biomol), or with the RXR ligand, 9-cis retinoic acid (Sigma Chemical Co), α-amanitin (Sigma Chemical Co), or ethanol vehicle (0.1%, vol:vol) in maintenance medium (MEMα with 4% FBS) for 24 hours unless indicated otherwise. Because 15d-PGJ2 can exert PPARγ-independent effects,28,29⇓ we used several ligands to strengthen our conclusions regarding PPARγ-dependent effects.
PPARγ and eNOS Expression
EC PPARγ expression was determined by measuring PPARγ 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 PPARγ (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 PPARγ 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.
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 PPARγ agonists. In separate studies to elicit overexpression of PPARγ, pCMXPPARγ32 (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 PPARγ expression in PPARγ- 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.
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.
PPARγ Activators Stimulate ·NO Release in EC
PPARγ expression was not altered in EC by either 15d-PGJ2 or ciglitazone treatment (data not shown). To determine if treatment with the PPARγ ligands causes functional activation of PAEC PPARγ, PAECs were transfected with pPPRE3tkluc followed by treatment with 15d-PGJ2 or ciglitazone for 24 hours. Luciferase activity was monitored and normalized to β-gal activity of a cotransfected pCMV-β-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 PPARγ-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 PPARγ 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 α-amanitin, a global RNA synthesis inhibitor, on ·NO release, in the absence or presence of 15d-PGJ2. Treatment with α-amanitin attenuated 15d-PGJ2–stimulated ·NO release to levels comparable with control treatment (Figure 2), indicating that PPARγ regulates ·NO release through changes in gene transcription.
To determine if PPARγ 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 PPARγ 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).
Effect of PPARγ Ligands on eNOS Expression
To determine if PPARγ 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 PPARγ-stimulated ·NO release is not attributable to increased eNOS or iNOS expression.
Effect of PPARγ Overexpression on Basal PAEC ·NO Release
To additionally confirm the role of PPARγ in regulating ·NO release, PAECs were transfected with the expression vector pCMXPPARγ. Western blot analysis of transfected cells confirmed that PPARγ expression was increased ≈3.5-fold over mock-transfected cells (Figures 6A and 6B). PPARγ overexpression in vascular endothelial cells significantly increased ·NO release with or without exogenous PPARγ ligands (10 μmol/L for 24 hours) compared with untreated mock-transfected cells (Figure 6C). These results additionally demonstrate that PPARγ plays a role in the enhancement of endothelial ·NO release.
The expression of PPARγ 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, PPARγ 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 PPARγ plays an important role in endothelial biology and the pathogenesis of vascular disease.
This report expands the recognized effects of PPARγ ligands on the vascular endothelium to include enhanced ·NO release and describes for the first time the expression of PPARγ in ECs derived from the pulmonary artery. However, similar stimulation of ·NO release by PPARγ 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 PPARγ 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⇓ PPARγ-induced stimulation of EC ·NO release provides a novel, and potentially unifying, mechanism for the direct vascular protective effects of PPARγ ligands, including thiazolidinedione medications. The mechanism by which PPARγ ligands lead to increased EC ·NO release remains to be defined. In contrast to our findings that PPARγ agonists enhance basal and ionophore-stimulated endothelial ·NO release, Ikeda et al41 demonstrated that PPARγ ligands inhibited IL-1β–stimulated ·NO levels in vascular smooth muscle cells. This effect was attributed to decreased iNOS expression meditated by suppression of nuclear factor (NF)-κB activity. However, in our study, iNOS was not detected in ECs treated with PPARγ activators (data not shown), suggesting that other iNOS-independent mechanisms account for the ability of PPARγ to modulate endothelial ·NO release.
Several lines of evidence suggest that the ability of PPARγ ligands to stimulate ·NO release does not represent simple nonspecific EC activation. For example, the PPARγ ligands used in this study, 15d-PGJ2 and ciglitazone, caused comparable increases in PAEC ·NO release but represent structurally diverse molecules that stimulated PPARγ functional activity to a comparable degree (Figure 1A) without altering PPARγ expression (data not shown). 15d-PGJ2 can exert PPARγ-independent effects, including inhibition of IκB kinase and interruption of NF-κB signaling.28,29⇓ Such PPARγ-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 α-amanitin. These findings indicate that structurally unrelated PPARγ 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 PPARγ-mediated effects42 suggests that 9-cis retinoic acid is acting, at least in part, through a PPARγ signaling pathway. Finally, EC ·NO release was stimulated not only by PPARγ ligands but also by overexpression of PPARγ. Control cells overexpressing PPARγ released greater levels of ·NO compared with their mock-transfected counterparts, suggesting that increased ·NO release was attributable to endogenous ligand. The inability of PPARγ ligands to stimulate additional increases in ·NO release from ECs overexpressing PPARγ (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 PPARγ 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 PPARγ 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 PPARγ ligands regulate the expression of other gene products, which, in turn, increase ·NO bioavailability. For example, PPARγ 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 PPARγ ligands increased EC Cu/Zn superoxide dismutase mRNA levels and decreased PMA-stimulated p47phox expression. Thus, PPARγ ligands could also potentially decrease EC superoxide levels to enhance ·NO bioavailability. Identification of the gene target involved in PPARγ-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 PPARγ 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 PPARγ-stimulated endothelial ·NO release may contribute to these vascular protective effects. In addition, our results demonstrate that PPARγ 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 PPARγ ligands to the prevention or treatment of vascular disease will require additional studies to better characterize the molecular targets regulated by PPARγ as well as their impact on clinical outcomes.
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; revision accepted September 18, 2002.
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