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Suppression of Pro-inflammatory Adhesion Molecules by PPAR-δ in Human Vascular Endothelial Cells

Originally published, Thrombosis, and Vascular Biology. 2008;28:315–321


Objective— Endothelial activation is implicated in atherogenesis and diabetes. The role of peroxisome proliferator-activated receptor-δ (PPAR-δ) in endothelial activation remains poorly understood. In this study, we investigated the anti-inflammatory effect of PPAR-δ and the mechanism involved.

Methods and Results— In human umbilical vein endothelial cells (HUVECs), the synthetic PPAR-δ ligands GW0742 and GW501516 significantly inhibited tumor necrosis factor (TNF)-α–induced expression of vascular cell adhesion molecule-1 and E-selectin (assayed by real-time RT-PCR and Northern blotting), as well as the ensuing endothelial-leukocyte adhesion. Activation of PPAR-δ upregulated the expression of antioxidant genes superoxide dismutase 1, catalase, and thioredoxin and decreased reactive oxygen species production in ECs. Chromatin immunoprecipitation assays showed that GW0742 switched the association of BCL-6, a transcription repressor, from PPAR-δ to the vascular cell adhesion molecule (VCAM)-1 promoter. Small interfering RNA reduced endogenous PPAR-δ expression but potentiated the suppressive effect of GW0742 on EC activation, which suggests that the nonliganded PPAR-δ may have an opposite effect.

Conclusions— We have demonstrated that ligand activation of PPAR-δ in ECs has a potent antiinflammatory effect, probably via a binary mechanism involving the induction of antioxidative genes and the release of nuclear corepressors. PPAR-δ agonists may have a potential for treating inflammatory diseases such as atherosclerosis and diabetes.

By using the selective agonists and siRNA-mediated gene silencing, we have demonstrated that ligand-activation of PPAR-δ in primary-cultured human endothelial cells has a potent antiinflammatory effect via a novel binary mechanism involving the induction of antioxidative genes and the release of nuclear corepressors.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. The PPAR family consists of 3 closely related subtypes: PPAR-α, β/δ, and γ.1 PPAR-δ is expressed ubiquitously, but its functions are largely unexplored. Saturated and polyunsaturated fatty acids are known to activate PPAR-δ,2–4 but physiological ligands of PPAR-δ have not been identified. Development of selective PPAR-δ ligands, including GW0742, GW501516, and L-165041, has extended the understanding of the physiological functions of this nuclear receptor. PPAR-δ may play a role in many important biological processes such as placental development,5 wound healing,6,7 oligodendrocyte differentiation,8,9 and carcinogenesis in the colon.10 Activation of PPAR-δ has been shown to raise high-density lipoprotein levels, reduce triglyceride levels, and improve insulin sensitivity.11 PPAR-δ increases fatty acid oxidation in skeletal muscle and ameliorates obesity and insulin resistance.12

The roles of PPAR-δ in atherosclerosis have been explored in mouse models of atherogenesis with the use of a loss-of-function approach and selective PPAR-δ agonists.13,14 The activation of endothelial cells (ECs), characterized by induced expression of pro-inflammatory adhesion molecules such as vascular cell adhesion molecule (VCAM)-1 and E-selectin, is an early step of atherosclerosis and often evoked by various atherogenic risk factors, including increased level of inflammatory cytokines (such as tumor necrosis factor-α [TNF-α] and interleukin [IL]-1), dyslipidemia, hyperinsulinemia, and insulin-resistant (type 2) diabetes.15 Recent studies have demonstrated that PPAR-δ is expressed in vascular smooth muscle cells as well as in ECs and plays potential roles in endothelial survival and proliferation.16–18 The expression of PPAR-δ is induced in response to pro-inflammatory cytokines such as TNF-α and platelet-derived growth factor (PDGF).16,19 However, the role of PPAR-δ in EC activation remains poorly understood. We aimed to elucidate the roles of PPAR-δ in TNF-α–induced expression of pro-inflammatory adhesion molecules and the ensuing EC-leukocyte interaction by using the synthetic PPAR-δ agonists GW0742 and GW501516 and knocking down the expression of endogenous PPAR-δ with small interfereing RNA (siRNA) in human vascular ECs.

Materials and Methods

Cells and Reagents

Human umbilical vein endothelial cells (HUVECs) were cultured as previously described.20 THP-1 was grown in RPMI 1640 containing 10% FBS. Bovine aortic endothelial cells (BAECs) were harvested from bovine aorta and maintained in DMEM with 10% FBS.21,22 Sources of reagents are listed in the supplemental materials (available online at

Northern Blotting

Fifteen μg of total RNA per lane was fractionated on formaldehyde-agarose gels, transferred to nylon membranes, and hybridized to cDNA probes for human VCAM-1, E-selectin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. The cDNA probes were synthesized by RT-PCR and labeled with 32P-dCTP as previously described.20 Results of autoradiography were scanned, and the signal intensity was analyzed by use of National Institutes of Health Image J software.

Real-Time Quantitative RT-PCR

Two μg of total RNA was reverse transcribed into cDNA with M-MLV reverse transcriptase and oligo-dT used as a primer. Real-time PCR involved SYBR-green dye and Taq polymerase. Please see supplemental materials for the primer sequences.

Immunoprecipitation and Western Blotting

Cellular proteins were extracted with lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 15 mmol/L EGTA, 100 mmol/L NaCl, 0.1% [wt/vol] Triton X-100 and complete protease inhibitor cocktail). The supernatants were immunoprecipitated with 1 μg of anti–BCL-6 antibody and protein A/G Plus-Agarose beads. The immunoprecipitates were immunoblotted as previously described.20 Cytoplasmic proteins were extracted with use of hypotonic lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5% NP-40). Nuclear proteins were extracted with use of high-salt buffer (20 mmol/L Tris-HCl, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 10% glycerol, 0.2 mmol/L EGTA).

Chromatin Immunoprecipitation (ChIP) Assay

Cells were cross-linked with 1% formaldehyde and quenched before harvest and sonication. The sheared chromatin was immunoprecipitated with anti–BCL-6 antibody (or control IgG) and protein A Sepharose beads. The eluted immunoprecipitates were digested with proteinase K, and DNA was extracted and underwent PCR with primers specific for the human VCAM-1 promoter region (−775 to −1015): (forward) 5′-CCAATGGGGGAGATAGACCT-3′ and (reverse) 5′-ACCGCAAACCCAGTTAAAAA-3′. The DNA samples were also analyzed using semi-quantitative real-time PCR and expressed as % of the input.

Plasmids, Transfection, and Reporter Assay

The PPRE-TK-luciferase reporter contains 3 copies of PPAR response elements (PPRE) from the acyl-coenzyme A (CoA) oxidase gene. pcDNA-PPAR-δ is the expression plasmid containing the full-length coding region of human PPAR-δ. Plasmids were transfected with the use of Lipofectamine 2000 (Invitrogen). The plasmid expressing β-galactosidase (pRSV-gal) was cotransfected to normalize the transfection efficiency. Cell lysates were harvested to measure luciferase and β-gal activities.

siRNA and Transfection

The siRNA targeting human PPAR-δ mRNA (NM_006238) was synthesized with the sense sequence of 5′-ACAGATGAAGACAGATGCACC as published.17 Double-strand RNA was transfected into HUVECs by use of Lipofectamine 2000. The siRNA against an irrelevant sequence derived from the Thermotoga maritimia genome was used as a control. The Cy3-labeled siRNA against luciferase mRNA (Dharmacon) was transfected and visualized under fluorescence microscopy.

Statistical Analysis

Quantitative data are expressed as mean±SEM. Statistical analyses involved Student t test or 1-way ANOVA followed by Newman-Keuls test. A P<0.05 was considered significant. Nonquantitative results were representative of at least 3 independent experiments.


PPAR-δ Inhibits the Expression of Pro-inflammatory Adhesion Molecules in ECs

To evaluate the ability of GW0742 to activate PPAR-δ in ECs, BAECs were transfected with pPPRE-TK-luc, together with pcDNA-PPAR-δ or the control vector. Promoter reporter assays showed that GW0742 activated PPAR-δ in ECs in a dose-dependent manner, with significant activation at 1 μmol/L (please see supplemental materials). To examine the effect of PPAR-δ on endothelial activation, HUVECs were pretreated with GW0742 (1 μmol/L, 24 hours), then TNF-α (2 ng/mL, 2 hours). As shown in Figure 1, the mRNA expression of pro-inflammatory adhesion molecules VCAM-1 and E-selectin was induced in response to TNF-α. GW0742 significantly inhibited the TNF-α–induced expression of VCAM-1 and E-selectin but not that of GAPDH gene. Similarly, another PPAR-δ agonist, GW501516, inhibited the TNF-α–induced expression of VCAM-1 and E-selectin. In addition, GW0742 and GW501516 significantly suppressed the induction of VCAM-1 and E-selectin stimulated by IL-1β (supplemental materials). To examine whether the PPAR-δ agonist decreased the TNF-α–induced mRNA level of VCAM-1 via an increase in mRNA turnover rate, HUVECs were pretreated with GW0742, stimulated with TNF-α, and treated with actinomycin (10 μg/mL, 2 hours), which inhibits RNA polymerase II. GW0742 did not decrease the half-life of VCAM-1 steady-state mRNA (supplemental materials), which indicates that the suppressive effect of GW0742 was probably via a transcriptional mechanism rather than a change in mRNA turnover.

Figure 1. GW0742 inhibited TNF-α–induced gene expression of adhesion molecules. HUVECs were pretreated with GW0742 (1 μmol/L) or GW501516 (0.1 μmol/L) for 24 hours and then incubated with TNF-α (2 ng/mL) or control medium for 2 hours. The RNA blots were hybridized for VCAM-1, E-selectin, and GAPDH. The bar graph shows the results from 3 independent experiments. *P<0.05, **P<0.01.

PPAR-δ Suppresses TNF-α–Induced Endothelial-Leukocyte Adhesion

EC-leukocyte adhesion assays were performed to determine whether GW0742 inhibits the recruitment of leukocytes to activated ECs. TNF-α markedly increased THP-1 adhesion to ECs, and GW0742 significantly suppressed the TNF-α–induced leukocyte adhesion (by 44.6±14.3% versus the solvent control, P<0.05; supplemental materials).

GW0742 Does Not Prevent NF-κB Nuclear Translocation in ECs

Because NF-κB plays a central role in the transcriptional regulation of adhesion molecule genes,23 we examined the effect of GW0742 on TNF-α–induced degradation of IκBα, the inhibitor of NF-κB, and the protein level of p65 NF-κB in cytosolic and nuclear portions. HUVECs were pretreated with GW0742 (1 μmol/L) or DMSO for 24 hours, then stimulated with TNF-α. Western blot analyses revealed that TNF-α induced IκBα degradation and the nuclear translocation of p65 NF-κB, which were not inhibited by GW0742 (Figure 2A). Furthermore, GW0742 did not inhibit TNF-α–induced phosphorylation of JNK, p38, and extracellular signal regulated kinase (ERK) (supplemental materials).

Figure 2. Effect of GW0742 on NF-κB activation. A, Western blots were detected with antibodies against IκBα, p65 NF-κB, α-tubulin, or histone H3. The bar graph shows the nuclear translocation of p65 expressed as percentage of nuclear (Nuc)/cytoplasmic (Cyto) contents. B, Immunoprecipitation was performed with anti–BCL-6, followed by Western blotting using anti–PPAR-δ or anti–BCL-6 antibody. C, ChIP assays were performed with anti–BCL-6 antibody and amplified with the primers for the VCAM-1 promoter. Bar graph represents the quantified data from 3 independent real-time PCR experiments. *P<0.05 vs control.

GW0742 Decreases the Association of BCL-6 With PPAR-δ in ECs

A previous study in 293 cells indicated that PPAR-δ, in the absence of a specific ligand, may bind BCL-6, a transcription repressor.14 Therefore, we performed coimmunoprecipitation (coIP) and ChIP experiments to explore this possibility in HUVECs. CoIP revealed the association of PPAR-δ with Bcl-6 (Figure 2B). GW0742 markedly reduced the amount of BCL-6 associated with PPAR-δ but not the amount of intracellular BCL-6 protein. In contrast, ChIP assays demonstrated that GW0742 increased the association of BCL-6 with the VCAM-1 promoter (Figure 2C), which suggests that GW0742 may suppress EC activation via triggering the relocation of BCL-6 from PPAR-δ to the promoter regions of the pro-inflammatory genes to inhibit their transcriptional activities.

GW0742 Attenuates TNF-α–Induced ROS via Upregulation of the Antioxidant Genes in ECs

Because increased generation of ROS is of crucial importance in EC activation,24 we further examined the effect of GW0742 on intracellular generation of ROS. HUVECs were pretreated with GW0742 for 24 hours, then stimulated with TNF-α for 1 hour. TNF-α induced a robust increase in ROS production, which was significantly inhibited by GW0742 (Figure 3A). To determine whether GW0742 reduced the TNF-α–induced increase in ROS by enforcing the antioxidative mechanism, we examined the expression of several key antioxidant genes. GW0742 significantly increased the expression of SOD1, catalase, and thioredoxin, by 72.8±29%, 31.9±10.5%, and 73±28.9%, respectively (Figure 3B).

Figure 3. GW0742 eliminated TNF-α–induced ROS production in ECs. HUVECs were treated with or without GW0742 for 24 hours, and stimulated with TNF-α for 1 hour. ROS was detected and expressed as fold induction. Gene expression was determined by qRT-PCR and expressed as fold induction after normalization to GAPDH. *P<0.05.

Effect of siRNA-Mediated Knockdown of PPAR-δ on Induction of Adhesion Molecules

To examine whether the antiinflammatory effect of PPAR-δ agonists depends on an adequate level of PPAR-δ expression, HUVECs were transfected with the PPAR-δ siRNA or control siRNA. The transfection efficiency of siRNA in HUVECs was confirmed with Cy3-labeled siRNA in that most cells showed uptake of siRNA (Figure 4A). PPAR-δ siRNA significantly decreased the mRNA level of endogenous PPAR-δ, by 75%, in ECs. Surprisingly, PPAR-δ siRNA not only did not inhibit the suppressive effects of the PPAR-δ ligand GW0742 on VCAM-1 and E-selectin induction but also enhanced these suppressive effects (Figure 4B and 4C). Thus, the synthetic ligand can robustly suppress the pro-inflammatory response in ECs at a reduced level of its endogenous cognate PPAR-δ, and the receptor, in the absence of specific ligands, may facilitate EC activation through physical association with anti-inflammatory corepressors such as BCL-6.

Figure 4. Effect of PPAR-δ siRNA on EC activation. HUVECs were transfected with PPAR-δ–specific or control siRNA for 48 hours before treatment with GW0742 or DMSO for 24 hours. Gene expressions were determined by qRT-PCR. *P<0.05; **P<0.01. The fluorescence photomicrograph shows uptake of Cy3-labeled siRNA in HUVECs.


Our results show that the PPAR-δ agonists GW0742 and GW501516 suppressed the TNF-α–induced expression of pro-inflammatory adhesion molecules VCAM-1 and E-selectin in ECs, as well as the ensuing leukocyte recruitment. We demonstrate for the first time that the suppressive effect on EC activation is associated with an antioxidative mechanism but does not prevent the nuclear translocation of NF-κB. Furthermore, knockdown of PPAR-δ potentiated the anti-inflammatory effect of PPAR-δ agonists in ECs.

Recent studies suggested a role of PPAR-δ in inflammatory processes and atherosclerosis. In macrophages, the PPAR-δ agonist GW0742 inhibited lipopolysaccharide (LPS)-induced expression of iNOS and COX2.14,25 However, in other cell types such as epithelial cells, eosinophils, neutrophils, and lymphocytes, the PPAR-δ agonist was ineffective in inhibiting inflammatory processes,26 which indicates that the effect is cell-type specific. GW0742 reduced atherosclerotic lesions and decreased the expression of MCP-1 and intercellular adhesion molecule-1 (ICAM-1) in the aorta of LDLR−/− mice.27 However, whether PPAR-δ agonists have a direct effect on endothelial activation, an initial step in atherogenesis, remains unclear. The effect of the PPAR-δ agonists GW0742 and GW501516 inhibiting TNF-α–induced expression of VCAM-1 and E-selectin in primary-cultured HUVECs and attenuating EC-leukocyte adhesion is PPAR-δ specific: both of these highly selective ligands for PPAR-δ exert potent effects when used at a concentration (0.1∼1μmol/L) lower than the EC50 for PPAR-α and -γ isoforms. In light of a critical role of EC activation in atherogenesis and type 2 diabetes, our finding is of importance in understanding the pathogeneses of these diseases. PPAR-δ activation, together with a lipid-modifying action11 and a role in protecting the survival of ECs,17 may have promising therapeutic application.

Excessive production of intracellular ROS in response to various stimuli (including TNF-α) can induce oxidative stress and has been implicated in the pathogenesis of cardiovascular and metabolic disorders, including atherosclerosis and diabetes.28,29,30 In this study we provide novel evidence that PPAR-δ agonists inhibited the TNF-α–induced ROS production in ECs. This finding is in agreement with a recent report describing a role of PPAR-δ in protecting against H2O2-induced apoptosis in ECs.17 In addition, we found that GW0742 increased the gene expression of SOD1, catalase, and thioredoxin, 3 important antioxidative molecules involved in the elimination of ROS.31 SODs convert superoxide anion into H2O2, which in turn can be reduced into water by catalase. Thioredoxin, via a series of coupled reactions, also functions as an antioxidant molecule in the cytosol of ECs.32 Coordinated induction of these key antioxidant genes can prevent intracellular accumulation of ROS and may account for the anti-inflammatory effect of PPAR-δ. Although the mRNA expression of SOD1 and catalase can be increased by ligands for PPAR-α and PPAR-γ,33–35 our results demonstrate for the first time a role of PPAR-δ in regulation of these antioxidant genes. Because functional PPREs have been found in the 5′-flanking regions of these genes,36 PPAR-δ may execute its antiinflammatory effect via direct activation of these antioxidant targets. Many transcription factors, including NF-κB, activator protein-1 and Ets-1, are known to be oxidant responsive and can modulate intracellular redox states.28,37 Because ROS activate complex signaling pathways,38–41 PPAR-δ may exert its antiinflammatory effect, at least in part, by positively regulating the antioxidant genes and eliminating excessive production of ROS.

The 5′-flanking regulatory regions of VCAM-1 and E-selectin genes lack PPRE. In this situation, PPAR-δ may downregulate these genes via trans-suppression of other transcription factors such as NF-κB, which plays an important role in regulating the mRNA expression of various pro-inflammatory adhesion molecules. In quiescent ECs, NF-κB is kept inactive by association with IκBα in the cytosol. On stimulation, IκB is phosphorylated and degraded, thus allowing subsequent nuclear translocation and activation of NF-κB. We found that GW0742, while inhibiting the inflammatory action of TNF-α, did not prevent the TNF-α–induced degradation of IκBα and subsequent nuclear translocation of NF-κB (Figure 2). EMSA results also showed no decrease in either basal or TNF-α–stimulated NF-κB DNA binding activity (data not shown). In addition, GW0742 showed no inhibitory effect on the TNF-α–stimulated activation of mitogen-activated protein kinase pathways in ECs. PPAR-δ probably interferes with transcription of the inflammatory genes via a corepressor-related mechanism at the chromatin level.

Another novel finding in this study is that the PPAR-δ agonist GW0742 potently inhibited EC activation, and the inhibitory effect not only persisted but was further enhanced, after the decrease of PPAR-δ expression by siRNA (Figure 4). In fact, knocking down PPAR-δ per se attenuated the induction of VCAM-1 and E-selectin in response to TNF-α (Figure 4B and 4C). Therefore, our findings suggest that the endogenous PPAR-δ, in the absence of specific ligands, may facilitate expression of these pro-inflammatory genes and the ligand robustly suppressed the pro-inflammatory response in ECs even when the level of its cognate receptor had been reduced. This seemingly paradoxical result agrees with observations for other members of the PPAR family such as PPAR-γ. Although the PPAR-γ agonists thiazolidinediones effectively sensitize insulin action, mice with heterozygous deficiency of PPAR-γ actually have improved insulin sensitivity. Unlike other PPAR isoforms, PPAR-δ is known to be a transcription repressor rather than an activator, when not liganded by specific agonists, through its ability to recruit potent repressors such as SMRT, SHARP, and class I histone deacetylases.42 A recent study showed that treatment with the PPAR-δ agonist GW501516 decreased the expression of pro-inflammatory cytokines MCP-1 and IL-1β in macrophages, and transplantation of bone marrow from the PPAR-δ-null mice also reduced atheroma formation in LDLR-null mice.14 To reconcile these observations, Lee and colleagues proposed an unconventional ligand-dependent transcriptional pathway whereby PPAR-δ controls an inflammatory switch through its association and dissociation with transcriptional repressors.14 In our study, we provide clear evidence that in ECs the ligand binding caused the dissociation of BCL-6 from PPAR-δ and the subsequent association of BCL-6 with the VCAM-1 promoter region (Figure 2B and 2C). Because the PPAR-δ agonist GW0742 also elicits a coordinated expression of a panel of antioxidant genes (Figure 3B), PPAR-δ agonists may suppress EC activation via a binary mechanism, that is, the synthetic ligand binds to PPAR-δ and recruits the coactivators to replace the corepressors such as BCL-6. The released corepressors relocate to repress the transcription of pro-inflammatory genes such as VCAM-1 and E-selectin and, thus, contribute to the vascular protection. In addition, the ligand-activated PPAR-δ is able to induce its target genes, among which are those encoding antioxidative enzymes (SOD1, catalase, and thioredoxin, etc), and to reduce the TNF-α–triggered oxidative stress. Such a synergistic action leads to a potent inhibition of endothelial activation and the ensuing leukocyte adhesion (Figure 5). This notion is supported by our siRNA results. Reduction of PPAR-δ abundance may also cause the translocation of the repressors from the PPAR-δ targets into the transcriptional complexes at the inflammatory genes. Because PPAR-δ is an abundantly expressed isoform in many types of tissues and cells (including ECs), even at a reduced level, the receptor would be still be sufficient to cause the transcription of its target genes in the presence of the specific agonist.

Figure 5. Proposed mode of action of PPAR-δ agonists in suppressing EC activation. PPAR-δ agonists suppress EC activation via a binary mechanism: (1) the agonists activate the PPAR-δ target genes encoding antioxidants, which reduces intracellular production of ROS stimulated by pro-inflammatory cytokines; (2) the agonists cause relocation of corepressors (CoR) to the pro-inflammatory genes. These effects act in concert to suppress EC activation.

In summary, we have demonstrated in HUVECs that PPAR-δ agonists have a strong anti-inflammatory effect potentiated by the siRNA-mediated reduction of endogenous PPAR-δ. PPAR-δ may be a potential therapeutic target for many cardiovascular and metabolic disorders such as atherosclerosis and diabetes, in which aberrant endothelial activation plays a significant pathophysiological role.

Original received June 14, 2007; final version accepted November 17, 2007.

Sources of Funding

This study was supported in part by grants from the Ministry of Education (PCSIRT) and National Science Foundation of China (#30470810 and 30670848 to N.W.), the Ministry of Science and Technology (2006CB503906 to N.W.), and the National Institutes of Health, USA (HL080518 to S.C.).




Correspondence to Nanping Wang, Institute of Cardiovascular Science, Peking University Health Science Center, Beijing 100083, China. E-mail


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