Cyclooxygenase-2-dependent oxidative stress mediates palmitate-induced impairment of endothelium-dependent relaxations in mouse arteries

Biochemical Pharmacology 91 (2014) 474–482

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Cyclooxygenase-2-dependent oxidative stress mediates palmitate-induced impairment of endothelium-dependent relaxations in mouse arteries Zhen Gao a,1, Huina Zhang a,b,1, Jian Liu a, Chi Wai Lau a, Pingsheng Liu b, Zhen Yu Chen c, Hung Kay Lee d, George L. Tipoe e, Hing Man Ho f, Xiaoqiang Yao a, Yu Huang a,* a

Institute of Vascular Medicine and Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong SAR, China National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China c School of Life Sciences, Chinese University of Hong Kong, Hong Kong SAR, China d Department of Chemistry, Chinese University of Hong Kong, Hong Kong SAR, China e Department of Anatomy, University of Hong Kong, Hong Kong SAR, China f School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 May 2014 Accepted 6 August 2014 Available online 19 August 2014

Palmitic acid, one of the saturated free fatty acids, impairs cardiovascular function as manifested by inducing vascular inflammation, apoptosis and over-production of reactive oxygen species (ROS) although the origin for ROS remains unclear. The present study investigated the cellular mechanisms underlying palmitate-induced impairment of endothelial function. Ex vivo treatment in tissue culture with palmitate concentration-dependently attenuated acetylcholine-induced endothelium-dependent relaxations, up-regulated the expression of cyclooxygenase-2 (COX-2) and elevated superoxide formation in mouse aortic endothelial cells (MAECs) measured by dihydroethidium (DHE) staining and electron paramagnetic resonance (EPR) spectroscopy. Superoxide scavengers, COX-2 inhibitor and thromboxane prostanoid (TP) receptor antagonist, but not COX-1 inhibitor reversed the harmful effects of palmitate. Furthermore, palmitate impaired acetylcholine-induced relaxations and raised superoxide in en face endothelium of aortas only from COX-1/ mice but not from COX-2/ mice. Palmitate increased the production and release of TXB2, a stable thromboxane A2 metabolite in mouse aortas, which was abolished by COX-2 inhibitor. Superoxide scavenger did not affect palmitate-induced upregulated expression of COX-2 in MAECs. Both real time PCR and luciferase reporter gene assay confirmed COX-2 up-regulation in palmitate-treated MAECs and NF-kB was substantially involved in this up-regulation. The present study provides novel evidence that palmitate up-regulates COX-2 through NF-kB-dependent mechanism and resultant COX-2-associated oxidative stress impairs endothelium-dependent relaxations in mouse aortas. ß 2014 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article: Sodium palmitate (PubChem CID: 2735111) Tempol (PubChem CID: 137994) Vitamin E (PubChem CID: 2116) SC-560 (PubChem CID: 4306515) Celecoxib (PubChem CID: 2662) S18886 (PubChem CID: 9938840) Dihydroethidium (PubChem CID: 128682) TEMPONE-H (PubChem CID: 98642) DMPO (PubChem CID: 1774) DTPA (PubChem CID: 3053) Acetylcholine (PubChem CID: 187) Phenylephrine (PubChem CID: 6041) L-NAME (PubChem CID 39836) Parthenolide (PubChem CID: 6473881) A23187 (PubChem CID: 11957499) Thromboxane B2 (Pubchem CID: 5283137) Hypoxanthine (Pubchem CID:790) Keywords: Palmitate Superoxide Cyclooxygenase-2 Endothelium-dependent relaxations

Abbreviations: ACh, acetylcholine; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; DHE, dihydroethidium; DMPO, 5,5-dimethyl-l-pyrroline-N-oxide; DTPA, diethylenetriaminepentaacetic acid; EDCFs, endothelium-derived contracting factors; EDR, endothelium-dependent relaxation; EPR, electron paramagnetic resonance; FFAs, free fatty acids; HX, hypoxanthine; MAECs, mouse aortic endothelial cells; NO, nitric oxide; PA, palmitate; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2a, prostaglandin F2a; PGI2, prostaglandin I2; PN, parthenolide; ROS, reactive oxygen species; SNP, sodium nitroprusside; TEMPONE-H, 1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidine hydrochloride; TP, thromboxane prostanoid; TXA2, thromboxane A2; XO, xanthine oxidase. * Corresponding author at: School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong SAR, China. Tel.: +852 3943 6787; fax: +852 26035022. E-mail address: [email protected] (Y. Huang). 1 Zhen Gao and Huina Zhang contributed equally to this work. http://dx.doi.org/10.1016/j.bcp.2014.08.009 0006-2952/ß 2014 Elsevier Inc. All rights reserved.

Z. Gao et al. / Biochemical Pharmacology 91 (2014) 474–482

1. Introduction Free fatty acids (FFAs) are elevated in the plasma of patients with metabolic syndrome [1,2] and they are likely to participate in vascular inflammation and contribute to the onset and progress of endothelial dysfunction. Palmitic acid (PA), a 16-carbon saturated FFA and one of the most common dietary fatty acids, triggers inflammation through activation of toll-like receptor-dependent IKKa-NF-kB signaling in human endothelial cells [3], increases endoplasmic reticulum stress and apoptosis in mouse endothelial cells [4], and promotes ROS formation from mitochondrial electron transport chain and NADPH oxidase in rat skeletal muscle [5]. In diabetic and obese individuals, increased saturated fatty acids stimulate prostaglandin production [6]. FFA activates protein kinase C and reduces endothelium-dependent relaxations in rat aortas [7] while inhibition of protein kinase C or adenosine monophosphate-activated kinase attenuates the stimulatory effect of PA on ROS production in human endothelial cells [8]. In addition, PA lowers nitric oxide (NO) bioavailability through reducing the phosphoryaltion of Akt and eNOS [9]. On the other hand, cyclooxyeganse-2 (COX-2), another inflammatory factor has emerged as an important contributor to vascular inflammation [10] and endothelial dysfunction in ageing and hypertension [11– 13]. Recent studies indicate that COX-2 is the downstream common target in response to pro-inflammatory factors such as angiotensin II and bone morphogenic protein 4 to induce and maintain endothelial dysfunction in hypertension and diabetes [14]. However, the positive functional association between palmitate and COX-2 in endothelial dysfunction is unclear despite several lines of conflicting reports in literature regarding such link in other cell types. For instance, saturated FFAs induce the expression of COX-2 through a toll-like receptor 4-dependent mechanism in monocytes and macrophages [15] while COX-2 inhibition augments the palmitic acid-mediated inflammation and insulin resistance in skeletal muscle [16]. Both palmitic acid and COX-2 are known to promote oxidative stress and vascular dysfunction [10,17–19], however, the exact role of ROS in the palmitic acid and COX-2 interaction remains unexplored in blood vessels. Against this background, we thus hypothesized that palmitate impairs endothelial function through COX-2 up-regulation and COX-2-dependent ROS over-production. 2. Materials and methods 2.1. Ethical approval The study was approved by the Chinese University of Hong Kong Animal Experimentation Ethics Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication, 8th Edition, 2011). 2.2. Preparation of palmitate Sodium palmitate (Sigma, USA) was prepared according to a method published [20]. Stock solution of palmitate was added to culture medium containing 10% fetal bovine serum (FBS, Gibco, USA) for cell treatment or organ culture. The same amount of solvent (ethanol) was added to culture medium as control. 2.3. Primary culture of mouse aortic endothelial cells (MAECs) Male C57BL/6 mice (4–6 weeks old) were supplied by Chinese University of Hong Kong Laboratory Animal Center. Mice were anesthetized with an intraperitoneal injection of a mixture of ketamine 10% (Alfasan, Netherlands) at dose of 50 mg/kg body

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weight plus xylazine 2% (Alfasan, Netherlands) at dose of 5 mg/kg body weight. Heparin (100 U/mL in PBS) was infused from the left ventricle into the circulation. The aorta was dissected out and cut into several ring segments that were incubated in collagenase type I-containing saline solution for 15 min at 37 8C and endothelial cells were collected by centrifugation and then re-suspended in 20% FBS-containing endothelial cell growth medium (EGM, Lonza, USA) [21]. MAECs were exposed to palmitate for 48 h in the absence and presence of one of the following inhibitors: tempol (superoxide scavenger, 100 mM), vitamin E (anti-oxidant, 30 mM), SC-560 (COX-1 inhibitor, 0.3 mM), celecoxib (COX-2 inhibitor, 3 mM) and S18886 (TP receptor antagonist, 0.1 mM). The concentration chosen for each inhibitor is reported to be specific for the designated target in literature [12–13,17]. Cultured mouse endothelial cells were verified using endothelial cell biomarker CD-144 (VE-Cadherin, Santa Cruz, USA) by flow cytometry assay which showed about 86.3% were endothelial cells (data not shown). H5V mouse endothelial cells were cultured in DMEM containing 10% FBS. When cells were in 90% confluent, the indicated plasmids were transfected by LipofectamineTM 2000 Reagent (Invitrogen, USA). 2.4. Measurement of superoxide by dihydroethidium staining and electron paramagnetic resonance spectroscopy Superoxide generation was determined on the Olympus Fluoview FV1000 laser scanning confocal system (Olympus, Tokyo, Japan). MAECs were incubated in dihydroethidium (DHE, 5 mM in ECM, Sigma–Aldrich, USA) for 30 min, and washed in ECM for three times to remove extracellular DHE before the fluorescence intensity was measured at emission wavelength of 520 nm. To further confirm superoxide generation in MAECs, electron paramagnetic resonances (EPR) spin trapping was utilized with 1-hydroxy-2,2, 6,6-tetramethyl-4-oxo-piperidine hydrochloride (TEMPONE-H, 100 mM, Alexis, Switzerland) and 5,5-dimethyl-lpyrroline-N-oxide (DMPO, Alexis, Switzerland) as spin trap agents [17]. All EPR samples were suspended in culture medium and collected in 200 ml glass tubes. Diethylenetniamine-pentaacetic acid (DTPA, 0.2 mM, Sigma–Aldrich, USA) was added to avoid reactions catalyzed by transition metals. EMX EPR spectrometer (Bruker, Germany) was used to measure X-band EPR spectra at room temperature. EPR settings were as follows: field center, 3480 G; field sweep, 100 G; microwave frequency, 9.746 GHz; microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.3 G; conversion time, 1024 ms; time constant, 640 ms. 2.5. Isometric force measurement Male C57BL/6 mice (10–12 weeks old) were supplied by Chinese University of Hong Kong while male COX-1/ and COX-2/  mice of C57BL/6 background were provided by University of Hong Kong. Mice were kept at 22–23 8C with a 12-h light/dark cycle and experiments were conducted according to institutional guidelines for the humane treatment of laboratory animals. Mice were euthanized by CO2 inhalation after an approval was obtained from the Animal Ethics Committee, Chinese University of Hong Kong. After mice were sacrificed, thoracic aortas were removed and cleaned of adhesive tissues in oxygenated ice-cold sterile PBS. Aortic rings (2 mm in length) were incubated in Dulbecco’s Modified Eagle’s Media (DMEM, Gibco, Gaithersberg, USA) supplemented with 10% FBS, plus 100 IU/ml penicillin and 100 mg/ml streptomycin, in a CO2 incubator with 95% O2 plus 5% CO2. Rings were exposed to palmitate with or without each of the following inhibitors: vitamin E, tempol, SC-560, celecoxib and

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S18886. After 48-h incubation, rings were suspended in myographs (Danish Myo Technology, Aarhus, Denmark) and changes in isometric tone were recorded. All rings were stretched to an optimal baseline tension (3 mN) and equilibrated for 1 h before they were contracted by 60 mM KCl. Endothelium-dependent relaxations were studied in phenylephrine (1 mM) pre-contracted endothelium-intact rings in response to acetylcholine. Endothelium-independent relaxations were also measured in response to sodium nitroprusside in some aortic rings. 2.6. Measurement of superoxide in en face endothelium of aortas from COX deficient mice Aortic rings isolated from COX-1/ and COX-2/ mice were incubated in DMEM supplemented with 10% FBS containing 100 IU/ml penicillin plus 100 mg/ml streptomycin, and they were kept in a CO2 incubator. Some rings were exposed to palmitate for 48 h, thereafter they were incubated for 30 min in DHE (5 mMcontaining medium (in mM: 121 NaCl, 5 NaHCO3, 10 Na-HEPES, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2 CaCl2, 10 glucose; pH 7.4)), washed three times with interval of 5 min, and then cut open longitudinally. The cut-open aortas were placed upside down between two coverslips with the endothelial layer facing upwards [22]. Images were taken at excitation wavelength of 515 nm and emission wavelength of 585 nm. Auto-fluorescence of elastin was taken at excitation wavelength of 488 nm and emission wavelength of 520 nm. 2.7. Western blotting Protein samples from aortas and MAECs homogenates were developed on 10% SDS–PAGE and transferred onto a PVDF membrane (Millipore Corp., Bedford, MA, USA). Nonspecific binding sites were blocked with 1% BSA in 0.05% Tween-20 TBS for 1 h. Then the membrane was incubated overnight at 4 8C with the primary antibodies: Anti-COX-2 (1:1000, Abcam, Cambridge, UK); Anti-COX-1 (1:1000, Caymen, Michigan, USA). Anti-GAPDH (1:5000, Ambion, Austin, TX, USA) was used as housekeeping protein. 2.8. Reverse transcription, real-time PCR Total RNA was extracted from MAEC using TRIzol reagent according to manufacturer’s protocol (Invitrogen, USA). 2 mg total RNA was used for the first-strand synthesis with iScriptTM cDNA Synthesis Kit (BIO-RAD, USA). Real-time PCR was used to analyze the cellular mRNA levels of COX-1, COX-2 and GAPDH. PCR analysis was performed using the following primers: for COX-1, sense 50 CAGGAGGTGTTTGGGTTGCT-30 , anti-sense 50 -TTCCCAGAGCCAGTATCCA-30 ; for COX-2, sense 50 -GGAAGTCTTTGGTCTGGTGCC-30 , anti-sense 50 -GTCTGCTGGTTTGGAAT AGTTGC-30 ; for GAPDH, sense 50 -AGGTCGGTGTGAACGGATTTG-30 , anti-sense 50 -TGTAGACCATGTAGTTGAGGTCA-30 . The SYBR Green PCR Master Mix (Invitrogen, USA) was employed for the amplifications reactions with a ViiA 7 Real-Time PCR Detection System (Invitrogen, USA). The fluorescence curves were analyzed using ViiA 7 RUO Software. 2.9. Luciferase reporter gene assay A series of pGL3-COX-2 plasmids containing different promoter truncated fragments (1900 bp, 432 bp, 275 bp, 192 bp) of COX-2 were kind gifts of Professor Manuel Fresno (Madrid, Spain). All constructs were confirmed by DNA sequencing. The purified luciferase plasmids were transiently transfected into H5V mouse endothelial cells using the LipofectamineTM 2000 Reagent at a ratio of 1 mL LipofectamineTM 2000 Reagent/0.5 mg of DNA according to

manufacturer’s instructions (Invitrogen, USA). The indicated reporter constructs (0.2 mg/well) were co-transfected with the internal control pRL-TK reporter (30 ng/well) to H5V cell line precultured in 24 well plate. Luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega, USA). 2.10. Measurement of TXB2 Mouse aortas were treated with 300 mM PA for 48 h in control and in the presence of 3 mM celecoxib. Thereafter, aortas were incubated with 100 mM L-NAME for 30-min, then transferred to centrifuge tubes that contained 200 mL bathing solution and 3 mM acetylcholine. Bathing medium was collected 3 min after tissue transfer for the assay of TXB2 (the stable metabolite of thromboxane A2) concentration using LC–MS/MS assay similar to previous report [23]. The corresponding protein concentration for each aortic ring was determined. The results were expressed as pg TXB2/ mg protein. As to LC–MS/MS assay, briefly, 200 mL of biological samples and TXB2 working solutions for standard curve were lyophilized by centrifugal vacuum concentrator (Savant DNA 120 SpeedVac Concentrator) to dryness. All the samples were redissolved by adding 30 mL of initial mobile phase (0.03% acetic acid: acetonitrile = 9:1). After centrifugation at 10,000  g for 10 min, the supernatant were transferred to sample vials for injection. Agilent 6460 Triple Quadrupole Mass Spectrometer with UHPLC (UHPLC–MS/MS, Agilent Technologies, Santa Clara, CA, USA) was employed in this study. The chromatographic separation was performed on an C18 column (2.1 mm  100 mm, 1.7 mm) (Waters, Milford, MA, USA) at 40 8C with the mobile phase consisting of 0.03% acetic acid in water (A) and acetonitrile (B) in a gradient elution method (10% of B from 0 to 1 min, 10–32% of B from 1 to 1.5 min, 32–38% of B from 1.5 to 8.5 min, 90% of B from 9 to 12.5 min, 10% of B from 12.6 to 18 min). The injection volume was 20 mL and the flow rate was 0.2 mL/min. The samples were kept at the auto-sampler at 4 8C throughout the analysis. The mass spectrometer was operated under negative ionization mode. The ESI source parameters were set as follows: drying gas temperature, 300 8C; gas flow, 8 L/min; nebulizer: 45 psi; sheath gas temperature: 350 8C; sheath gas flow: 10 L/min; capillary voltage: 3500 V, and nozzle voltage: 1000 V. For the detection of TXB2, the mass spectrometer was operated in multiple reaction monitoring (MRM) mode and the transition from m/z 369.2 to m/z 195.1 was monitored, the fragmentor voltage and collision energy were 120 V and 10 eV respectively. 2.11. Drugs Acetylcholine, phenylephrine and sodium nitroprusside (Sigma–Aldrich, St Louis, MO, USA) were dissolved in water; palmitate (Sigma–Aldrich, St Louis, MO, USA) was dissolved in ethanol; celecoxib (Pfizer, USA), SC-560, vitamin E, tempol, L-NAME (NGnitro-L-arginine methyl ester, Sigma–Aldrich, St Louis, MO, USA), S18886 and parthenolide (Cayman, Michigan, USA) were dissolved in DMSO. 2.12. Data analysis Data represent means  SEM of n rings from different mice. Concentration–response curves were analyzed by nonlinear regression curve fitting using GraphPad Prism 4.0 software (Version 4.0). Protein expression was quantified by Quantity One software (Bio-Rad laboratories Int., USA) and normalized to GAPDH. Student’s t-test (two-tailed) was used when two groups of means were compared. One-way ANOVA followed by the Bonferroni post hoc test were used when more than two treatments were compared. p < 0.05 indicates statistical difference between groups.

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3. Results 3.1. Palmitate increases superoxide production and induces endothelial dysfunction Exposure (48 h) to palmitate stimulated superoxide rise in cultured mouse aortic endothelial cells (MAECs) in a concentration-dependent manner (Fig. 1A and B) while the solvent ethanol had no effect (data not shown). Incubation with palmitate concentration-dependently impaired acetylcholine (ACh)-induced endothelium-dependent relaxations in mouse aortas (Fig. 1C) without affecting endothelium-independent relaxations to sodium nitroprusside (data not shown), while other two fatty acid oleic acid and arachidonic acid had no effect on endothelial function (data not shown). Based on clinical materials and previous studies, the plasma concentration of free palmitic acid in diabetic patients and in diabetic db/db mice is up to 300 mM [2,24,25], we therefore chose 300 mM palmitate to treat endothelial cells and aortas in the following experiments. 3.2. Superoxide scavenger and antioxidant reverse palmitatestimulated superoxide production and endothelial dysfunction Palmitate (300 mM)-stimulated superoxide generation in MAECs was reversed by superoxide scavenger tempol (100 mM) or antioxidant vitamin E (30 mM) (Fig. 2A and B). To further confirm palmitate-induced superoxide elevation, the EPR spectroscopy was performed to detect production of superoxide anions. Palmitate at 300 mM indeed triggered superoxide rise in MAECs, which was largely attenuated by vitamin E (Fig. 2A–D). Superoxide-generating reaction of hypoxanthine plus xanthine oxidase served as positive control. Neither tempol nor vitamin E alone modulated ACh-induced relaxations but treatment with each of them restored palmitate-impaired relaxations (Fig. 2E), suggesting

Fig. 1. PA stimulates superoxide anion generation and impairs ACh-induced endothelium-dependent relaxations in C57BL/6 mouse aortas. (A) PA (48-h incubation) concentration-dependently increased superoxide anion production in primary mouse aortic endothelial cells (MAECs) detected by DHE fluorescence dye. (B) Summarized data for DHE fluorescence signal intensity. (C) Ex vivo treatment with PA (48-h) concentration-dependently attenuated ACh-induced relaxations in mouse aortas. Results are means  SEM of five separate experiments. * p < 0.05 vs. Control.

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that superoxide anions are likely to mediate palmitate-induced endothelial dysfunction. 3.3. Inhibition of COX-2 and TP receptor reverses palmitatestimulated superoxide production In MAECs, palmitate (300 mM) stimulated superoxide production, which was inhibited by COX-2 inhibitor celecoxib (3 mM) and thromboxane prostanoid (TP) receptor antagonist S18886 (0.3 mM), but not by COX-1 inhibitor SC-560 (Fig. 3A–D), suggesting that palmitate-stimulated superoxide production is most likely to be secondary to COX-2 activation. 3.4. Palmitate stimulates COX-2-dependent superoxide production To verify the critical role of COX-2 in endothelial cell superoxide generation, aortas from both COX-1/ and COX-2/ mice were studied. Palmitate treatment caused a marked increase in superoxide accumulation in en face endothelium of aortas from wild type and COX-1/ mice but not from COX-2/ mice (Fig. 4A and B). 3.5. Inhibition of COX-2 and TP receptor improves endothelial function in palmitate-treated mouse aortas Both celecoxib and S18886 inhibited palmitate-stimulated COX-2-dependent superoxide generation in endothelial cells

Fig. 2. Tempol and vitamin E reverse PA-stimulated superoxide production and endothelial dysfunction. (A–D) Elevated superoxide production in PA (300 mM)treated MAECs was reversed by co-incubation with vitamin E (30 mM) or tempol (100 mM) as detected by DHE staining (A and B) and by EPR spectroscopy (C and D) with HX XO (100 mM hypoxanthine plus 0.01 units/ml xanthine oxidase) serving as positive control. (E) PA (300 mM)-induced impairment of ACh relaxations was inhibited by co-treatment with vitamin E (30 mM) or tempol (100 mM). Results are means  SEM of five separate experiments. *p < 0.05 vs. Control; #p < 0.05 vs. PA.

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Fig. 3. COX-2 inhibitor and TP receptor antagonist reverse PA-stimulated superoxide production. PA (300 mM)-stimulated superoxide production in MAECs was normalized by co-incubation with COX-2 inhibitor (3 mM) and TP receptor antagonist S18886 (0.1 mM), but not COX-1 inhibitor SC-560 (0.3 mM) as shown by using EPR spectroscopy (A and B) and DHE staining (C and D). HX XO  served as positive control. Results are means  SEM of five experiments. p < 0.05 vs. # Control; p < 0.05 vs. PA.

Fig. 4. PA increases ROS generation in en face endothelium of mouse aortas. (A) Representative images and (B) summarized data showing the intracellular superoxide production by DHE staining in en face endothelium of aortas from control wild type (WT), COX-1/ and COX-2/ mice with and without 48-h exposure to PA (300 mM). Red: DHE fluorescence (excitation wavelength: 515 nm). Green: auto-fluorescence of elastin underneath endothelium (excitation wavelength: 488 nm). Results are means  SEM of aortas from five mice. *p < 0.05 between control and PA. Calibration bar: 100 mm.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

(Fig. 3), suggesting that COX-2-derived arachidonic acid metabolites might mediate palmitate-stimulated superoxide increase leading to endothelial dysfunction. As expected, co-treatment with either COX-2 inhibitor celecoxib or TP receptor antagonist S18886 but not COX-1 inhibitor SC-560 rescued palmitate-induced impairment of ACh-induced relaxations in aortas of wild type mice (Fig. 5A). Individual inhibitor alone had no effect (Fig. 5A). To confirm the positive role of COX-2, both COX-2 and COX-1 deficient mice were used (Fig. 5D). Palmitate was still able to impair AChinduced relaxations in aortas only from COX-1/ mice (Fig. 5B) but not in those from COX-2/ mice (Fig. 5C). 3.6. TP receptor-mediated superoxide overproduction and endothelial dysfunction The TP receptor antagonist S18886 reduced palmitate-stimulated superoxide production and palmitate-induced impairment of endothelium-dependent relaxations, suggesting a harmful effect of TP receptor activation. We then tested whether TP receptor agonist U46619 (a thromboxane A2 mimic) can stimulate superoxide production. Indeed, U46619 (100 nM) elevated superoxide generation in MAECs and this effect was reversed by S18886 (Fig. 6A and B). To further demonstrate the functional role of the TP receptor in superoxide production, U46619 (100 nM) was used to treat mouse aortas for 24 h and the results showed that U46619 attenuated ACh-induced relaxations and this attenuation was again reversed by S18886 and superoxide scavenger tempol, but not by celecoxib (Fig. 6C). In addition, a combined treatment with S18886 plus

Fig. 5. Effects of COX-2 inhibitor, TP receptor antagonist, and COX-2 knockout on PA-impaired relaxations in mouse aortas. (A) PA (300 mM)-induced impairment of ACh-induced relaxations was inhibited by co-treatment with celecoxib (3 mM) and S18886 (0.1 mM), but not by SC-560 (0.3 mM). PA attenuated Ach-induced relaxations in aortas from COX-1/ mice (B) but not in those from COX-2/ mice (C). (D) The expression of COX-1 and COX-2 in aortas from COX-1/ and COX-2/ mice. Results are means  SEM of five mice. *p < 0.05 vs. Control; #p < 0.05 vs. PA.

tempol did not produce an additive effect, suggesting that palmitate-stimulated superoxide elevation is likely associated with TP receptor activation (Fig. 6D). Finally, the result of LC–MS/ MS assay showed that PA at 300 mM increased the production of

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suppressed COX-2 promoter activity induced by palmitate (Fig. 7D), indicating that NF-kB binding motifs are likely to play an important role in the regulation of palmitate-stimulated COX-2 expression. By contrast, palmitate did not affect the luciferase activity of COX-1 (Fig. 7C). 4. Discussion

Fig. 6. TP receptor-mediated superoxide production is involved in endothelial dysfunction. (A and B) DHE staining showing that U46619 (100 nM, 24 h) increased ROS generation in MAECs, which was inhibited by co-incubation with S18886 (0.1 mM), tempol (100 mM) but not by celecoxib (3 mM). (C) Treatment (24 h) with U46619 (100 nM) attenuated ACh-induced relaxations in mouse aortas, which was reversed by co-treatment with S18886 and tempol but not with celecoxib. (D) For comparison, S18886 and tempol reversed PA-induced impairment of relaxations, whereas S18886 plus tempol did not produce additive effect. (E) PA (300 mM, 48 h) increased generation of TXB2 (the stable metabolic product of thromboxane A2), which was reversed by 3 mM celecoxib in mouse aortas. Results are means  SEM of five mice. *p < 0.05 vs. Control; #p < 0.05 vs. U46619 or PA.

TXB2, the stable metabolite of thromboxane A2 in mouse aortas and this increase was reversed by co-treatment with 3 mM celecoxib (Fig. 6E). 3.7. Palmitate increases COX-2 expression through NF-kB-dependent mechanism We next sought to determine whether palmitate regulated COX-2 expression. Palmitate significantly increased the expression of COX-2 but not COX-1 at both protein (Fig. 7A) and mRNA (Fig. 7B) levels. In order to investigate the transcriptional regulation of COX-2 by palmitate and its possible mechanism, luciferase reporter vectors driven by COX-2 promoters were transiently transfected into H5V mouse endothelial cells. Palmitate dramatically enhanced the promoter activity of pGL3-COX-2-1900 in both cell lines as well as the positive control pGL3-NF-kB-Luc (which contains six tandem NF-kB binding sites upstream luciferase gene), and NF-kB inhibitor parthenolide (20 mM) reversed the effect of palmitate, indicating that NF-kB possibly mediates the palmitate-induced up-regulation of COX-2 (Fig. 7C). To further confirm this possibility, luciferase reporter vectors controlled by a series of truncated promoters of COX-2 (192, 275, 432 and 1900) were used. The deletion of NF-kB binding sites (388 to 380 bp and 223 to 214 bp) significantly

The present study demonstrates that superoxide anions, COX-2, and TP receptors play important roles in palmitate-induced impairment of endothelium-dependent relaxations. Superoxide scavengers, COX-2 inhibitor, and TP receptor antagonist all reverse the impaired relaxations and superoxide over-generation in palmitate-treated mouse aortas. More importantly, both COX-2/  and COX-1/ mice were used to explore the link between palmitate and COX-2 up-regulation in relation to superoxide and endothelial function. In addition, the present study also shows that palmitate up-regulates the expression of COX-2 partially through NF-kB-dependent mechanism and COX-2 up-regulation is the upstream of superoxide generation, which contributes to impairment of endothelium-dependent relaxations (Fig. 8). Arachidonic acid released from plasma membranes by phospholipases can be readily oxygenated by COX to form PGH2; the latter is the common precursor for the formation of five vasoactive prostanoid products, e.g., PGI2, PGE2, PGD2, PGF2a and TXA2 via respective synthases [26,27] and the competitive enzymatic interactions determine the relative components of PG synthesis pathways [28]. Both COX-1 and COX-2 present in blood vessels can catalyze the production of endothelium-derived contracting factors (EDCFs) [13,19,29–31]. COX-1-derived endoperoxides in endothelium contract vascular smooth muscle through activating the TP receptor [30], while endothelium-derived COX-2-derived PGF2a also activates the TP receptor to mediate endotheliumdependent contractions in arteries of hamsters and rats [11,13]. The present study shows that palmitate-mediated attenuation of ACh-induced relaxations involves both COX-2 and TP receptors as the impaired relaxations are largely rescued by treatment with selective COX-2 inhibitor (celecoxib) and TP receptor antagonist (S18886). This is further confirmed by the use of COX-2-deficient mice in which palmitate no longer inhibits endothelium-dependent relaxations. By contrast, the involvement of COX-1 appears to be negligible because palmitate is still able to attenuate relaxations in aortas of COX-1 knockout mice and palmitate-induced impairment in wild type mouse aortas is unaffected by COX-1 inhibitor SC-560. Furthermore, U46619, the stable TXA2 mimic attenuated endothelium-dependent relaxations in mouse aortas and this effect was antagonized by S18886 but not by COX-2 inhibitor, suggesting that COX-2-derived contracting prostanoid is the likely mediator of palmitate-induced endothelial dysfunction. Indeed, palmitate stimulates the production and release of TXA2 (the natural agonist for TP receptors) as reflected by its stable metabolite TXB2 in mouse aortas and this effect is abolished by COX-2 inhibitor celecoxib. Superoxide triggers the release of contracting prostanoids such as TXA2 and PGF2a via the action of COX-2 in high glucose-treated human aortic endothelial cells and in hypertensive rat arteries [11,32]. Superoxide is also involved in PGD2 generation in LPStreated macrophages [33], while ROS is unrelated to COX-2mediated production of EDCFs in hamster aortas [13]. The present study shows that co-treatment with superoxide scavenger tempol or anti-oxidant vitamin E largely restores the impaired relaxations in palmitate-treated mouse aortas and vitamin E scavenges palmitate-stimulated superoxide in mouse aortic endothelial cells. In addition, co-treatment with tempol plus S18886 did not result in additional benefit to improve relaxations, suggesting that superoxide, COX-2 and TP receptor are likely to operate along the same

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Fig. 7. PA increases expression of COX-2 through NF-kB-dependent mechanism. (A) PA increased the protein expression of COX-2 but not COX-1 in MAECs. Tempol did not affect PA-induced COX-2 up-regulation. (B) PA elevated COX-2 mRNA level in MAECs in a concentration-dependent manner. (C) The promoter activity of COX-2 but not COX-1 was up-regulated by PA, which was abolished by NF-kB inhibitor parthenolide (PN, 20 mM) in H5V mouse endothelial cells. pGL-NF-kB-Luc acted as positive control. Results are means  SEM of five experiments. *p < 0.05 vs. Control; #p < 0.05 vs. PA. (D) PA enhanced the promoter activity of pGL3-COX-2-1900 and pGL3-COX-2-432, which was significantly diminished when the COX-2 promoter sequence was deleted to 275 bp. The deletion of COX-2 promoter sequence to 192 bp diminished PA-induced luciferase activity. Results are means  SEM of four experiments. *p < 0.05 vs. Control; #p < 0.05 vs. PA.

signaling axis leading to endothelial dysfunction in aortas exposed to palmitate. However, little is known about the relation between superoxide and COX-2 in palmitate-treated arteries despite palmitate was shown before to stimulate superoxide production in endothelial and vascular smooth muscle cells [34]. The present study with the use of COX deficient mice provides more definite evidence that palmitate up-regulates COX-2 expression in endothelial cells and subsequently elevated superoxide impairs endothelium-dependent relaxations. Palmitate-stimulated superoxide rise can be reversed by both celecoxib and S18886 but not by SC-560, and palmitate is still able to trigger superxide production in endothelium en face of aortas from COX-1 but not from COX-2 knockout mice, thus supporting a critical role of COX-2-derived

prostanoids in superoxide over-production in palmitate-treated endothelial cells and aortas. In addition, COX-2 inhibitor celecoxib also reverses the palmitate-induced reduction of NO production in HUVECs (n = 4, data not shown). Indeed, U46619, the thromboxane A2 mimic also stimulates superoxide rise in MAECs and this rise is abolished by co-treatment with S18886 or tempol but not by celecoxib. This result is in line with the functional observation that S18886 and tempol but not celecoxib inhibit U46619-induced reduction of ACh relaxations in mouse aortas. Taken together, it is probable that palmitate up-regulates the expression and activity of COX-2 to augment the release of prostanoids such as TXA2; the latter elevates superoxide anions in endothelial cells to lower NO bioavailability and thus impairs endothelial function.

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Madrid, Spain) for pGL3-COX-2 promoter plasmids. This study was supported by National Natural Science Foundation of China (81270932), National Basic Research Program of China (2012CB517805), Beijing Natural Science Foundation (5122028), Hong Kong Scholarship Program (XJ2011045), CRF Grants from Research Grants Council of Hong Kong (CUHK2/CRF/12G), and CUHK High Promise Initiatives. References

Fig. 8. The proposed mechanism by which PA up-regulates COX-2 through NF-kBdependent mechanism and subsequent COX-2-derived prostanoids act as both autocrine and paracrine agonists to stimulate TP receptors, resulting in ROS overproduction in endothelial cells, thus lowering NO bioavailability and impairing endothelial function.

We finally confirmed that palmitate actually increased the COX-2 expression at both protein and mRNA levels in a concentrationdependent manner without affecting COX-1 expression. These results agree with the functional observations that inhibition of COX2 but not COX-1 ameliorates the palmitate-induced endothelial dysfunction. We then elucidated that the palmitate-induced COX-2 expression occurred at the transcriptional level using a luciferase reporter gene assay. Palmitate indeed enhanced the promoter activity of COX-2 but not COX-1, and NF-kB inhibitor parthenolide largely diminished palmitate-induced COX-2 promoter activity. Further experiments with transient transfection of COX-2 promoter truncated luciferase reporter plasmids showed that NF-kB motifs were likely to play a significant role in the regulation of palmitateinduced COX-2 expression. Previous studies described that palmitate stimulated NF-kB transcriptional activity through activating TLR4 signaling pathway, which induced inflammatory responses in several types of cells, including macrophages, endothelial cells, adipocytes and even skeletal muscle cells [3,16,35,36]. However, in the latter study, inhibiting COX-2 activity exacerbates instead protects against palmitate-triggered inflammatory reaction and insulin resistance in skeletal muscle. Taken together with our results, it is probable that palmitate-induced increase in COX-2 expression or activity may have different functional impacts depending on the type of cells or tissues under examination. In summary, the present study reveals a cellular mechanism in endothelial cells that links palmitate stimulation and subsequent up-regulation of COX-2 and COX-2-dependent superoxide generation in impairing endothelium-dependent relaxations in mouse aortas (Fig. 8). Saturated FFAs such as palmitate are positively associated with elevated cardiovascular risks and insulin resistance in patients [37,38] and endothelial dysfunction is a common pathological change evolving into major cardiovascular and renal complications in these patients. The present study demonstrates the effectiveness of either COX-2 inhibitor or TP receptor antagonist to inhibit the palmitate-induced oxidative stress and to restore endothelial dysfunction, suggesting a potential therapeutic value of these inhibitors to ameliorate diabetic vasculopathy related to high levels of FFAs. Conflicts of interest None to declare. Acknowledgments We are grateful to Prof. Manuel Fresno (Centro de Biologı´a Molecular ‘‘Severo Ochoa’’, CSIC-UAM, Universidad Auto´noma,

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