Published online before print July 14, 2005, doi: 10.1161/01.ATV.0000177805.65864.d4
© 2005 American Heart Association, Inc.
Vascular Biology |
Peroxisome Proliferator-Activated Receptor 
Ligands Stimulate Endothelial Nitric Oxide Production Through Distinct Peroxisome Proliferator-Activated Receptor –Dependent Mechanisms
From the Department of Medicine, Atlanta Veterans Affairs and Emory University Medical Centers, Atlanta, Ga.
Correspondence to John A. Polikandriotis, PhD, Atlanta VAMC (151-P), 1670 Clairmont Rd, Decatur, GA 30033. E-mail jpolika{at}emory.edu
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Abstract |
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Objective— We recently reported that the peroxisome proliferator-activated receptor
(PPAR) ligands 15-deoxy-
12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone increased cultured endothelial cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS). The current study was designed to characterize further the molecular mechanisms underlying PPAR-ligand–stimulated increases in endothelial cell NO production.
Methods and Results— Treating human umbilical vein endothelial cells (HUVEC) with PPAR ligands (10 µmol/L 15d-PGJ2, ciglitazone, or rosiglitazone) for 24 hours increased NOS activity and NO release. In selected studies, HUVEC were treated with PPAR ligands and with the PPAR antagonist GW9662 (2 µmol/L), which fully inhibited stimulation of a luciferase reporter gene, or with small interfering RNA to PPAR, which reduced HUVEC PPAR expression. Treatment with either small interfering RNA to PPAR or GW9662 inhibited 15d-PGJ2-, ciglitazone-, and rosiglitazone-induced increases in endothelial cell NO release. Rosiglitazone and 15d-PGJ2, but not ciglitazone, increased heat shock protein 90-eNOS interaction and eNOS ser1177 phosphorylation. The heat shock protein 90 inhibitor geldanamycin attenuated 15d-PGJ2- and rosiglitazone-stimulated NOS activity and NO production.
Conclusions— These findings further clarify mechanisms involved in PPAR-stimulated endothelial cell NO release and emphasize that individual ligands exert their effects through distinct PPAR-dependent mechanisms.
This study characterizes the molecular mechanisms underlying peroxisome proliferator-activated receptor (PPAR) ligand–stimulated increases in endothelial nitric oxide production. The data indicate that different PPAR ligands increase endothelial cell nitric oxide production by distinct PPAR-dependent signaling pathways that could represent novel targets for pharmacological intervention in vascular disease.
Key Words: peroxisome proliferator-activated receptor • nitric oxide • endothelium • endothelial nitric oxide synthase • thiazolidinedione
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Introduction |
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Endothelium-derived nitric oxide (NO) is a key molecule in vascular biology that decreases vascular tone, smooth muscle cell proliferation, leukocyte adhesion, and platelet aggregation.1–6 Endothelial dysfunction, characterized by impaired endothelial NO production, participates in the pathogenesis of atherosclerotic disease and is associated with risk factors for vascular disease, including hypercholesterolemia, diabetes mellitus, insulin resistance, and obesity.7 Our recent studies demonstrate that the peroxisome proliferator-activated receptor
(PPAR) ligands 15-deoxy-12,14-PG J2 (15d-PGJ2) and ciglitazone stimulate NO release from endothelial cells (ECs).8 Understanding the mechanisms of PPAR ligand-induced stimulation of EC NO release may provide novel insights into the vascular protective effects of PPAR ligands. In ECs, type III endothelial nitric oxide synthase (eNOS) produces NO from the amino acid L-arginine. eNOS is regulated not only at the level of expression,9–11 but also post-translationally by mechanisms including interactions of eNOS with other proteins12–15 and eNOS phosphorylation.16–19 For example, specific stimuli including vascular endothelial growth factor (VEGF), histamine, and shear stress have been shown to activate eNOS by promoting the interaction of eNOS with heat shock protein 90 (hsp90), a molecular chaperone protein.14 Hsp90 has been shown to increase eNOS activity by (1) recruiting Akt, the serine protein kinase B, to phosphorylate eNOS at ser1177,20 (2) facilitating the displacement of eNOS from inhibitory interactions with caveolin,21 and (3) increasing the affinity of eNOS for calmodulin.22 In addition to protein-protein interactions, several specific sites of phosphorylation also regulate eNOS activity. For example, phosphorylation of eNOS at ser1177 increases electron flux from the reductase to the oxygenase domain of eNOS and also increases enzyme activity.16,23
Our laboratory recently demonstrated that (1) overexpression of PPAR or treatment with 9-cis retinoic acid, the ligand for the PPAR heterodimer RXR, enhanced EC NO release, (2) the PPAR ligands 15d-PGJ2 and ciglitazone, without altering PPAR expression, stimulated a PPAR response element-luciferase reporter construct in transfected ECs and significantly increased basal as well as calcium ionophore-induced endothelial NO release, and (3) neither 15d-PGJ2 nor ciglitazone altered eNOS mRNA levels, whereas 15d-PGJ2, but not ciglitazone, decreased eNOS protein expression.8 These findings led us to further characterize the molecular mechanisms underlying the ability of PPAR ligands to stimulate endothelial NO production. The current study demonstrates that 15d-PGJ2, ciglitazone, and rosiglitazone increase NO release in ECs by distinct PPAR-dependent signaling pathways.
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Methods |
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HUVEC Treatment Protocols
HUVECs were grown on 100-mm dishes or 6-well plates and maintained in endothelial cell growth media according to the protocols provided by the manufacturer (Clonetics). When 90% confluent, HUVECs were treated with an equal volume of vehicle, 5 µmol/L A23187 (Alexis Biochemicals), or with the PPAR
ligands, 10 µmol/L 15d-PGJ2 (Calbiochem), 10 µmol/L ciglitazone (Biomol Research Laboratories), or 10 µmol/L rosiglitazone (Cayman Chemicals) for 24 hours at 37°C in a 5% CO2 incubator, a concentration previously shown to increase HUVEC NO release.8 In selected experiments, HUVECs were also cotreated for 24 hours with the PPAR antagonist GW9662 (2 µmol/L; Cayman Chemicals), which covalently modifies Cys285 in helix 3 of the PPAR ligand-binding domain,24 or the hsp90 inhibitor geldanamycin (GA) (1 µmol/L; Calbiochem). All treatments were prepared as a stock solution in water, ethanol, or dimethyl sulfoxide, and diluted in endothelial cell growth media to their final concentrations. Control conditions included HUVECs treated with vehicle alone.
Analysis of HUVEC NO Release and NOS Activity
NO release from HUVEC was determined by subjecting culture media from treated monolayers to chemiluminescence analysis with a Sievers Nitric Oxide Analyzer as previously reported.8,25 Total NOS activity was quantified by measuring conversion of [3H]-L-arginine to [3H]-L-citrulline by the Nitric Oxide Synthase Assay Kit (Calbiochem) as previously reported.26
Transfection Protocols
Following protocols provided by the manufacturer, HUVECs (50% confluence) were transfected with optimized concentrations of either human PPAR small interfering RNA (siRNA) Cat. # sc-29455), control nonsense fluorescein conjugate siRNA (Cat. # sc-36869), or with mock conditions using siRNA transfection reagent alone (Santa Cruz Biotechnology, Inc). Forty-eight hours after transfection, cells were treated with PPAR ligands. Whole cell lysates were subjected to immunoblotting with anti-PPAR antibodies (Santa Cruz Biotechnology, Inc) to confirm small interfering RNA to PPAR-induced alterations in PPAR expression. In separate studies, HUVECs were transfected with a PPAR reporter vector containing 3 PPAR response elements (PPREs) in which luciferase expression is induced by PPAR agonists, or with the control vector (Panomics, Inc), using the FuGENE 6 transfection reagent (Roche). Twelve hours after transfection, HUVECs were treated with PPAR ligands for 24 hours. Cells were then harvested and luminescence was measured in a microplate luminometer.
Western Blotting and Immunoprecipitation Techniques
After treatment and washing, HUVEC monolayers were collected into lysis buffer (20 mmol/L Tris pH 7.4, 2.5 mmol/L EDTA, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 100 mmol/L NaCl, 10 mmol/L NaF, 1 mmol/L Na3VO4, and anti-protease cocktail pill), sonicated, and centrifuged. Each sample (20 µg protein) was subjected to SDS-PAGE (4% to 12% gradient gels) (Invitrogen), and proteins were transferred to polyvinylidene fluoride membranes and immunoblotted with primary antibodies (1:1000) to phospho-eNOS at ser1177 (Cell Signaling), hsp90 (Stressgene), or eNOS (BD Transduction Laboratories) in TBS-T (10 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.1% Tween) containing 5% powdered non-fat dry milk or 3% bovine serum albumin for the phospho-antibody. Hsp90-eNOS interactions were examined by incubating whole cell lysates (200 µg protein) with 5 µg monoclonal eNOS antibody overnight at 4°C on a rocker. Antibody-eNOS complexes were collected by incubation with GammaBind sepharose beads (Amersham Pharmacia), and the immunocomplexes were precipitated by centrifugation. Immunoprecipitated proteins were then separated with SDS-PAGE, transferred to polyvinylidene fluoride membranes, and immunoblotted for eNOS and hsp90. Protein bands were identified with chemiluminescence and quantified with laser densitometry.
Statistical Analysis
Overall treatment effects were examined by ANOVA. Post hoc analysis to detect differences between specific groups was accomplished with the Student Neuman Keuls test. The level of statistical significance was taken as P<0.05.
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Results |
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We have previously reported that treatment with PPAR
agonists increased NO release from human umbilical vein or aortic endothelial cells without increasing PPAR or eNOS expression.8 To verify that 15d-PGJ2, ciglitazone, and rosiglitazone increased endothelial NO release through PPAR-dependent signaling, HUVECs were transfected with PPAR-specific siRNA and then assayed for PPAR expression and NO release in the presence of these PPAR ligands. Western blotting verified that the expression of PPAR was specifically and significantly reduced by the cognate PPAR siRNA duplex compared with mock transfections or transfections with a control fluorescein conjugate-scrambled siRNA whose sequence is unrelated to PPAR (Figure 1A and 1B). Importantly, compared with mock transfections, PPAR siRNA significantly reduced 15d-PGJ2-, ciglitazone-, and rosiglitazone-dependent NO release (Figure 1C).
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By transfecting HUVECs with a luciferase reporter gene containing 3 PPREs as an index of PPAR transactivation, Figure 2A demonstrates that the PPAR antagonist GW9662 (2 µmol/L) fully prevented PPAR transactivation caused by 15d-PGJ2, ciglitazone, or rosiglitazone. Furthermore, treating HUVECs with 2 µmol/L GW9662 significantly reduced 15d-PGJ2-, ciglitazone-, and rosiglitazone-dependent NO release (Figure 2B). In contrast, GW9662 failed to inhibit NO release stimulated by the calcium ionophore A23187, indicating that GW9662 specifically inhibits PPAR ligand-stimulated NO release. The Toxilight bioassay kit (Cambrex), which measures the release of adenylate kinase from damaged cells, was used to demonstrate that the inhibitory effects of neither PPAR siRNA nor GW9662 were attributable to cell injury (data not shown).
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Because PPAR ligands increased EC NO release without increasing eNOS expression, post-translational mechanisms of eNOS regulation were examined. As illustrated in Figure 3A and 3B, treatment with 15d-PGJ2, but not ciglitazone or rosiglitazone, significantly increased the overall cellular content of hsp90 when normalized to actin expression and decreased eNOS expression (Figure 3C), as previously reported.8 In contrast to 15d-PGJ2-mediated increases in NO release (Figure 2B), the PPAR antagonist GW9662 had no effect on 15d-PGJ2-mediated increases in hsp90 expression (Figure 3B) or decreases in eNOS expression (Figure 3C). Importantly, neither the PPAR ligands nor the antagonist GW9662 had significant effects on actin expression. Treatment with 15d-PGJ2 and rosiglitazone, but not ciglitazone, increased the amount of hsp90 associated with eNOS, and the PPAR antagonist GW9662 attenuated these increases in hsp90-eNOS interactions (Figure 4). The importance of hsp90-eNOS interactions in 15d-PGJ2- and rosiglitazone-stimulated HUVEC NO release is further supported by the demonstration that the hsp90 inhibitor GA completely abolished the increase in NO production seen after 15d-PGJ2 and rosiglitazone treatments (Figure 5A), whereas GA had no effect on ciglitazone-stimulated NO production (Figure I, available online at http://atvb.ahajournals.org). Similarly, 15d-PGJ2- and rosiglitazone-stimulated eNOS activity was also inhibited by GA treatment (Figure 5B).
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Because hsp90-eNOS interaction can recruit kinases that phosphorylate eNOS, eNOS phosphorylation after treatment with PPAR ligands was examined. Compared with treatment with vehicle alone, 15d-PGJ2 and rosiglitazone, but not ciglitazone, significantly increased the phosphorylation of eNOS at ser1177, an effect attenuated by the PPAR antagonist GW9662. None of the PPAR ligands examined caused significant alterations in the phosphorylation of eNOS at thr495 (data not shown).
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Discussion |
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The molecular action of the thiazolidinedione (TZD) class of insulin-sensitizing medications, currently used with patients with type 2 diabetes, involves direct activation of the PPAR
receptor.27 PPARs are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. PPAR is expressed at low levels in many tissues, where its activation produces diverse tissue-specific effects. In the vessel wall, PPAR is expressed in smooth muscle28 and endothelial cells.29,30 Current evidence suggests that activation of PPAR exerts beneficial effects on the vasculature. For example, studies in non-diabetic mouse models of atherosclerosis demonstrated that TZDs reduced lesion formation.31–33 TZD therapy has also been associated with improved endothelial function,34–37 reduced carotid intimal thickening,38 and neointimal formation after coronary stent placement.39 The vascular protective effect of PPAR ligands in humans was recently extended to non-diabetic subjects with documented coronary disease wherein rosiglitazone reduced common carotid arterial intima-media thickness progression.40 Taken together, these reports indicate that PPAR activation modulates the biology of the vascular wall through mechanisms that are incompletely defined.
Several studies have demonstrated that PPAR ligands exert direct effects on vascular wall cells in vitro. Troglitizone increased endothelial NO release through both PPAR-dependent and -independent signaling pathways involving differential eNOS phosphorylation and alterations in VEGF and VEGF receptor expression,41 confirming previous evidence that 15d-PGJ2 and ciglitazone increased EC NO release.8 The current study extends these reports by demonstrating that rosiglitazone also increased EC NO production. Neither 15d-PGJ2, ciglitazone, nor rosiglitazone, however, altered the expression of VEGF or its receptor in the current study (Figure II, available online at http://atvb.ahajournals.org). In addition, 10 µmol/L 15d-PGJ2 was previously reported to lower glutathione levels and cell viability in cultured HUVECs,42,43 effects not observed in the current model (data not shown). These apparent discrepancies in endothelial response to various PPAR ligands may relate to differences between studies in culture conditions that modulate PPAR effects, such as culture media serum concentrations.43 The current study was therefore designed to examine several PPAR ligands under identical culture conditions to determine whether individual PPAR ligands exert their effects on endothelial NO production through common pathways.
The most important findings in the current study are that under identical culture conditions, the same concentration of 3 different PPAR ligands activated EC PPAR and increased EC NO release to a comparable degree (Figures 1 and 2
). Furthermore, the ability of each PPAR ligand to stimulate EC NO release was PPAR-dependent because it was inhibited by treatment with either siRNA or the PPAR antagonist GW9662. These findings demonstrate that the current model represents an appropriate system to examine potential ligand-specific mechanisms of PPAR-induced alterations in endothelial NO production.
The current study also provides novel evidence that selected PPAR ligands modulate EC NO release through hsp90-related mechanisms. Hsp90 increases eNOS activity and NO release in a dose-dependent manner.14,44–47 Of the PPAR ligands studied, only 15d-PGJ2 increased overall HUVEC hsp90 expression (Figure 3), an effect that was not blocked by GW9662, suggesting a PPAR-independent mechanism.48–50 Although the hsp90 gene has not been reported to contain a PPRE, cyclopentenone prostaglandins similar to 15d-PGJ2 stimulated heat shock protein expression through activation of heat shock transcription factor,51 providing a plausible explanation for the ability 15d-PGJ2 but not ciglitazone or rosiglitazone to increase hsp90 expression. Treatment with 15d-PGJ2 or rosiglitazone, but not ciglitazone, however, increased hsp90 binding to eNOS (Figure 4) suggesting a potential role for this protein-protein interaction in enhanced NO production. The role of hsp90 was further supported by studies using the hsp90 inhibitor GA. As previously reported for VEGF- and bradykinin-induced NO release,52,53 GA attenuated 15d-PGJ2- or rosiglitazone-stimulated NO release (Figure 5). Previous reports, as well as our studies (data not shown), indicate that GA can redox cycle and generate superoxide, which could impair NO release through a mechanism independent of its effects on hsp90.54 GA not only reduced 15d-PGJ2 and rosiglitazone-stimulated NO release, however, but it also inhibited 15d-PGJ2- and rosiglitazone-mediated increases in NOS activity (Figure 5A). Taken together, these data suggest an important role for hsp90 in 15d-PGJ2- and rosiglitazone-stimulated eNOS activity and NO production.
Hsp90 could activate eNOS activity and increase NO production through recruitment of kinases to phosphorylate eNOS and through displacement of eNOS from inhibitory interactions with caveolin. Treatment with PPAR ligands reduced eNOS-caveolin interactions consistent with eNOS activation (Figure III, available online at http://atvb.ahajournals.org). Furthermore, treatment with 15d-PGJ2 and rosiglitazone, but not ciglitazone, also increased eNOS ser1177 phosphorylation (Figure 6), a post-translational modification facilitated by hsp90 and associated with enhanced enzyme activity.23,47 Although the dephosphorylation of eNOS at thr495 can also increase enzyme activity, neither 15d-PGJ2, ciglitazone, nor rosiglitazone altered eNOS thr495 phosphorylation (data not shown). The precise mechanism of 15d-PGJ2- and rosiglitazone-induced eNOS phosphorylation remains to be defined but could involve enhanced kinase activity, reduced phosphatase activity, or both. Several kinase pathways are known to phosphorylate eNOS at ser1177 including protein kinase B, extracellular signal regulated kinase, AMP-activated protein kinases, calmodulin-dependent kinase II, protein kinase G, and protein kinase A.44,55 To date, we have determined that wortmannin, a specific inhibitor of PI-3 kinase/ protein kinase B signaling, does not attenuate PPAR ligand-mediated NO production (data not shown). Additional mechanistic studies are currently ongoing in our laboratory to identify upstream signaling pathways activated by PPAR ligands that promote eNOS-hsp90 interaction and eNOS phosphorylation.
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Although 15d-PGJ2, ciglitazone, and rosiglitazone each stimulated EC NO release and caused comparable degrees of PPAR activation, ciglitazone, unlike the other PPAR ligands, failed to stimulate hsp90 expression, eNOS-hsp90 association, or eNOS ser1177 phosphorylation. The mechanisms of ciglitazone-stimulated NO release continue to be defined, but are likely attributable in part to ciglitazone-induced reductions in the expression of endothelial NADPH oxidase components and superoxide generation, as well as increased superoxide dismutase expression.56 By decreasing superoxide levels, PPAR ligands can reduce superoxide-mediated interactions with NO to increase NO release and enhance NO bioavailability. These studies demonstrate that PPAR ligands regulate several pathways involved in endothelial NO production and bioavailability.
Although PPAR-independent effects of specific ligands48–50 could potentially account for differing biological effects of PPAR ligands, the current study demonstrates that PPAR-ligand–stimulated NO release from HUVECs was fully inhibited by either PPAR siRNA (Figure 1) or GW9662 (Figure 2). Therefore, we speculate that these ligand-specific effects are attributable to characteristics of the PPAR receptor itself. PPAR receptor activation involves not only ligand binding but also the association or dissociation of coactivator and corepressor complexes.57–59 Activation of PPAR involves ligand-specific conformational changes in the receptor that recruit distinct populations of coactivator and corepressor proteins to induce ligand-specific patterns of gene expression. These considerations suggest that whereas PPAR activation in vascular endothelial cells may control an overall pattern of gene expression that promotes NO production, the biological effect of individual ligands may be mediated through discreet pathways.
In summary, our findings demonstrate that activation of PPAR in vascular endothelial cells provides a novel mechanism for stimulating endothelial NO release. 15d-PGJ2, ciglitazone, and rosiglitazone increased NO production by distinct signaling pathways that are PPAR-dependent. These results provide further evidence that PPAR ligands have the potential to directly modify vascular endothelial function and to modulate the production of NO, a critical mediator in maintenance of normal vascular physiology. These findings further refine our understanding of novel targets for pharmacological intervention in vascular disease and contribute to the definition of the molecular targets for TZDs and related PPAR ligands in the vasculature.
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Acknowledgments |
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This work was supported by grants from the Veterans Affairs Research Service (C.M.H.) and the National Institutes of Health (DK 61274).
Received January 20, 2005; accepted June 27, 2005.
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