Oleanolic acid ameliorates high glucose-induced endothelial dysfunction via PPARδ activation

Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA) is a pentacyclic triterpenes widely distributed in food, medicinal plants and nutritional supplements. OA exhibits various pharmacological properties, such as hepatoprotective and anti-tumor effects. In this study, we analyzed the effect of OA on endothelial dysfunction induced by high glucose in human vascular endothelial cells (ECs). Western blotting showed that OA attenuated high glucose-reduced nitric production oxide (NO) as well as Akt-Ser473 and eNOS-Ser1177 phosphorylation in cultured human umbilical vein ECs (HUVECs). Next, luciferase reporter assay showed that OA activated peroxisome proliferators-activated receptor δ (PPARδ) activity. Quantitative reverse transcriptase PCR (qRT-PCR) demonstrated that OA increased the expressions of PPARδ target genes (PDK4, ADRP and ANGPTL4) in ECs. Meanwhile, the induced expressions of PDK4, ADRP and ANGPTL4 by OA were inhibited by GSK0660, a specific antagonist of PPARδ. In addition, inhibition of PPARδ abolished OA-induced the Akt-Ser473 and eNOS-Ser1177 phosphorylation, and NO production. Finally, by using Multi Myograph System, we showed that OA prevented high glucose-impaired vasodilation. This protective effect on vasodilation was inhibited in aortic rings pretreated with GSK0660. Collectively, we demonstrated that OA improved high glucose-impaired endothelial function via a PPARδ-mediated mechanism and through eNOS/Akt/NO pathway.

OA attenuated the high glucose-induced impairment of Akt-Ser 473 and eNOS-Ser 1177 phosphorylation. Phosphorylation of Akt at Ser 473 plays a critical role in the transduction of insulin signaling and, in turn, phosphorylates its downstream substrate eNOS at Ser 1177 to increase eNOS activity 13,19 . In HUVECs, HG treatment suppressed the phosphorylation of both Akt-Ser 473 and eNOS-Ser 1177 . However, OA attenuated the suppression of Akt and eNOS phosphorylation by HG ( Fig. 2a and b). As shown in Fig. 2c and d, OA could increase the basal levels of Akt-Ser 473 and eNOS-Ser 1177 phosphorylation.

OA activated PPARδ in ECs.
We previously demonstrated a protective effect of PPARδ on endothelial function in diabetic mice through activating the Akt/eNOS signaling pathway 17 . Thus, we examined whether OA activated PPARδ in ECs. BAECs were transfected with the PPRE-driven luciferase reporter and PPARδ expression plasmid before exposed to OA. The reporter assay showed that OA increased the luciferase activity of PPARδ (Fig. 3a). Further, we examined the effects of OA on the expressions of the endogenous PPARδ target genes including pyruvate dehydrogenase kinase 4 (PDK4), adipose differentiation-related protein (ADRP) and angiopoietin-like protein 4 (ANGPTL4). As shown in Fig. 3b, the mRNA expressions of PDK4, ADRP and ANGPTL4, were increased by OA. In addition, pretreatment with GSK0660, a selective antagonist of PPARδ , effectively abrogated the induction of PDK4, ADRP and ANGPTL4 by OA (Fig. 3c), suggesting a PPARδ -specific mechanism.
OA attenuated the high glucose-induced impairment of NO production via PPARδ. In order to study whether PPARδ activity is required for the protective effect of OA on NO production against high glucose, HUVECs were pretreated with a selective PPARδ antagonist GSK0660 and then with OA (10 μ M, 12 h) before the exposure to high glucose. The results demonstrated that, in the presence of GSK0660, OA failed to restore the high glucose impaired NO production (Fig. 4a). We further examined the role of PPARδ in the Akt and eNOS (b) HUVECs and BAECs were treated with indicated concentrations of OA for 24 h, and cell viability was measured by MTT. All data were expressed as mean ± SEM of triplicate experiments. (c) BAECs were preincubated with or without OA (10 μ M) for 12 h, then, treated with high glucose (HG, 30 mM, 12 h), mannitol served as vehicle control to HG. NO was detected by using DAF-FM diacetate (40 × objective).
Scientific RepoRts | 7:40237 | DOI: 10.1038/srep40237 phosphorylation induced by OA. As shown in Fig. 4b and c, inhibition of PPARδ activity by GSK0660 significantly diminished the OA-induced eNOS-Ser 1177 and Akt-Ser 473 phosphorylation. In the presence of GSK0660, OA also failed to protect ECs against the high glucose-impaired phosphorylations of Akt and eNOS ( Fig. 4d and e).
OA enhanced endothelium-dependent vasodilatation via PPARδ. Compared with the time-matched vehicle control, OA elicited concentration-dependent relaxations pre-contracted by KCl or phenylephrine in the rat arterial segments ( Fig. 5a and b). Next, aortic rings were exposed to high glucose co-incubated with or without OA for 12 h. Acetylcholine-induced endothelium-dependent vasorelaxation was impaired by high glucose (Fig. 5c). HG impaired blood vessel relaxation was attenuated by OA. However, the effect of OA was abolished when the arteries were pre-incubation with GSK0660 (Fig. 5d). These results suggest that OA improves endothelial relaxation and attenuates the high glucose impairment via the PPARδ activation.

Discussion
In this study, we have demonstrated for the first time that OA, as a natural product, can ameliorate the high glucose-triggered endothelial function by activating the nuclear receptor PPARδ . Previous studies showed that pentacyclic triterpenes from fruits and plants could increase estrogen receptor activities 20 and synthetic triterpenes has been identified as ligands for the PPARγ 21 . In addition, reporter gene assays showed that oleanolic acid increased PPARα activity in spontaneously transformed keratinocyte cell line HaCaT and CV-1 cells 22 . Punica granatum flower (PGF) and its component oleanolic acid enhanced the PPARα reporter activity in human embryonic kidney 293 cells, and this effect was completely suppressed by a selective PPARα antagonist 23,24 . We and others have previously demonstrated that synthetic PPARδ agonists such as GW501516 and GW0742 could ameliorate a number of pathological features associated with diabetes and metabolic syndrome. In ECs, these agonists inhibited the expression of pro-inflammatory genes 14 . In mouse models for atherosclerosis, synthetic agonists of PPARδ reduced atherosclerotic lesions 25 . In obese diabetic mice, the PPARδ agonists prevented fatty liver by repressing sterol regulatory element binding protein-1 (SREBP1) and improved vascular function 17,26 . These results clearly indicate that PPARδ is a promising target for treating metabolic syndrome and related cardiovascular diseases. However, clinical uses of such potent synthetic agonists have been seriously hindered largely due to the claimed cardiovascular adverse effects of PPARγ 27 . Therefore, searching natural compounds with PPARδ modulating properties and, in particular, vascular protective activity, represent a promising approach for metabolic syndrome and related vascular disorder, also in view of their advantageous effects in endothelial system and obesity prevention 28 .
Emerging evidence revealed the beneficial effects of OA on endothelial function in vascular disorders associated to metabolic diseases. OA induced vasodilatation in isolated aortas from both normotensive and spontaneously hypertensive rats 5,29 . NO was involved in the vasodilator responses of OA, and in ECs, OA induced phosphorylation of eNOS-Ser 1177 to increase NO production 30 . Further studies have shown that OA increased the phosphorylation of Akt-Ser 473 and led to eNOS activation 31 . These observations were largely consistent with our results. However, the mechanisms by which OA regulates Akt/eNOS pathway were poorly understood. Herein, we demonstrated that OA exerts the endothelial protective effects mainly by PPARδ activation. This notion was supported by its capacity of activating the PPAR-reporter (Fig. 3a) and the induction of multiple endogenous PPARδ target genes, PDK4, ANGPL4 and ADRP (Fig. 3b). More importantly, the effects of OA were attenuated by a selective PPARδ antagonist GSK0660 (Fig. 3c). It remains to be examined whether this pentacyclic triterpenoid serves as a bona fide ligand for PPARδ or enhances the transcriptional activity of PPARδ by a post-translational modification.
It is well known that exposure to high glucose impairs endothelial functions and decreases the NO production. These deleterious effects are attributed to the inhibition of expression and/or activity of eNOS 32,33 . PPARδ agonists could ameliorate the endothelium-dependent vasodilatation impaired by HG via activating PI3K/Akt/ eNOS pathway in MAECs 17 . In the present study, we found that OA protected endothelial cells from high glucose injury (Fig. 1). This effect could also be inhibited by blocking PPARδ (Fig. 2). In endothelium-specific PPARδ deficient mice, both endothelium-dependent relaxations to ACh and endothelium independent relaxations to the NO donor were significantly impaired in the arteries, accompanied by decreased eNOS-Ser 1177 phosphorylation 34 . It is suggested that OA restores HG-impaired endothelial function by a PPARδ -mediated activation of Akt/eNOS signaling pathways. It is worth noting that OA and PPARδ both possess an anti-oxidative property 35 . Under oxidative stress, eNOS undergoes uncoupling and produces superoxide rather than NO. GTP cyclohydrolase I (GTPCH I), a rate-limiting enzyme responsible for tetrahydrobiopterin (BH4) de novo synthesis, switches eNOS from uncoupled to coupled state to balance the NO and reactive oxygen species (ROS) generations 36 . GTPCH I protein level was up-regulated by GW501516, as well as the production of BH4 in endothelial progenitor cells 37 . Thus, the effects of OA on GTPCH I expression and eNOS coupling remain to be investigated.
In summary, this study provided evidence that OA, a component of pentacyclic triterpenes, is a natural modulator of PPARδ and ameliorates the high glucose impaired endothelial dysfunction. It is suggested that OA has a potential application in the treatment of diabetes related vascular diseases.

Ethics statement. The animal experiment was approved by the institutional review board of Xi'an Jiaotong
University and performed in accordance with the institutional and national guidelines for the care and use of animals.  MTT Assay for Cell Viability. Cell viability was evaluated by MTT assay. HUVECs and BAECs were seeded in 96-well plates and cultured until 80% confluence. Then the cells were treated with the indicated concentrations of OA for 24 h before incubation with 5 mg/ml MTT at 37 °C in 5% CO 2 atmosphere for 4 h. Next, the culture medium was removed and the formazan formed in the reaction was dissolved in 150 μ l DMSO. Cell viability was presented as a percentage of the vehicle.

Reagents
Quantitative Reverse Transcriptase-PCR (qRT-PCR). Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), converted to cDNA by iScript cDNA synthesis kit (Bio-rad, Hercules, CA). Real-time PCR was performed by using SYBR Green Supermixes (Bio-rad) and a 7500 Real-time PCR machine (Applied Biosystems, Foster City, CA). The reaction conditions consisted of: stage 1, 95 °C for 10 min; stage 2, 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, which were concluded by the melting curve analysis process. Fold changes of gene expression were calculated using the 2 −ΔΔCt method. The primer sequences are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Plasmids, Transfection and Reporter Assay. The plasmids expressing PPARδ and PPRE-TK-luciferase
reporter contains 3 copies of PPAR-response elements (PPRE) from the acyl-coenzyme A (CoA) oxidase gene were transfected into subconfluent BAECs together with pRSV-gal, a plasmid expressing β -galactosidase by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). 24 hours later, the cells were treated with or without OA. Cell lysates were harvested to measure the luciferase and β -gal activities.
Detection of NO. NO release was measured with using DAF-FM diacetate. Briefly, BAECs seeded on glass coverslips and treated with different stimuli. By the end of treatment, the cells were incubated with DMEM containing DAF-FM (5 μ M) for 30 min in the dark at 37 °C and then washed with PBS. Images were obtained using fluorescence microscopy (Olympus America Inc., NY, USA).
Vascular reactivity. Aortic rings (3 mm) were dissected from Sprague-Dawley rats and incubated with different treatments at 37 °C in 5% CO 2 , then mounted in Multi Myograph System (Danish Myo Technology A/S, Denmark) to measure the isometric force. During the whole experiment, the solution was continuously oxygenated with a gas mixture of 95% O 2 plus 5% CO 2 . To determine the vasodilatory effect of OA, KCl (60 mM) or phenylephrine (Phe, 10 μ M) was used to constrict arterial rings in advance. After sustained contraction was obtained, the concentration-dependent responses of OA (0.01-100 μ M) were examined. In order to evaluate the role of high glucose, arterial rings were incubated in medium containing 30 mM glucose in the presence or absence of 10 μ M OA to examine the acetylcholine-induced vasodilation.
Statistical analysis. Quantitative data are expressed as mean ± SEM. Student's t test and ANOVA were used to analyze the statistical significance for the differences between two or among more groups, respectively. The dose response curves were analyzed by using two-way ANOVA followed by Bonferroni post-tests. P < 0.05 was considered significant. Non-quantitative results were representative of at least three independent experiments.