Angiotensin II-Induced Oxidative Stress in Human Endothelial Cells

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Article Cite This: Chem. Res. Toxicol. 2019, 32, 1412−1422

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Angiotensin II-Induced Oxidative Stress in Human Endothelial Cells: Modification of Cellular Molecules through Lipid Peroxidation Seon Hwa Lee,*,† Shuhei Fujioka,†,‡ Ryo Takahashi,†,§ and Tomoyuki Oe*,† †

Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan

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ABSTRACT: Angiotensin (Ang) II is a major bioactive peptide of the renin/angiotensin system and is involved in various cardiovascular functions and diseases. Ang II type 1 (AT1) receptor mediates most of the physiological effects of Ang II. Previous studies have revealed that the lipid peroxidation products 4-oxo-2(E)-nonenal (ONE) and 4hydroxy-2(E)-nonenal (HNE) readily modify the N-terminus and Asp1, Arg2, and His6 residues of Ang II, and these modifications alter the biological activities of Ang II. Ang II is known to stimulate the formation of reactive oxygen species (ROS) that mediate cardiovascular remodeling. Another major consequence of ROS-derived damage is lipid peroxidation, which generates genotoxic aldehydes such as ONE and HNE. This study demonstrated that Ang II induced lipid peroxidation-derived modifications of cellular molecules in EA.hy926 cells, a human vascular endothelial cell line. Ang P (ONE- and ROS-derived N-terminal pyruvamide Ang II) and [His6(HNE)]-Ang II were detected in the medium of EA.hy926 cells incubated with Ang II, and their concentrations increased dose-dependently upon the addition of ascorbic acid (AscA) and CuSO4. Cells were then subjected to metabolic labeling using SILFAC (stable isotope labeling by fatty acids in cell culture) with [13C18]-linoleic acid. Analysis of cellular phospholipids indicated over 90% labeling. [13C9]-Thiadiazabicyclo-ONE-glutathione adduct as well as Ang P and [His6([13C9]-HNE)]-Ang II was detected in the labeled cells upon treatment with Ang II and their concentrations increased in an Ang II dose-dependent manner. Incubation of the labeled cells with losartan, an AT1 receptor blocker, inhibited the formation of modified Ang IIs in a dose-dependent manner. These results indicate that Ang II induces lipid peroxidation and modification of various cellular molecules and these reactions are mediated by the activation of AT1 receptor. Therefore, lipid peroxidation could be one mechanism by which Ang II contributes to cardiovascular dysfunction.

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INTRODUCTION Accumulating studies have implicated oxidative stress as a substantial contributor to the age-related degenerative diseases,1 cancer,2 and cardiovascular disease.3 Oxidative stress can induce the generation of reactive oxygen species (ROS) and the activation of enzymes such as cyclooxygenases (COXs) and lipoxygenases (LOXs). ROS exhibit high reactivity toward polyunsaturated fatty acids (PUFAs) in lipids, resulting in the formation of PUFA hydroperoxides.4 COXs and LOXs can convert linoleic acid (LA) and arachidonic acid (AA) to the corresponding hydroperoxides,4 which undergo transition metal ion- and L-ascorbic acid (AscA)-dependent decomposition to form the reactive aldehydes 4-oxo-2(E)-nonenal (ONE), 4-hydroxy-2(E)-nonenal (HNE), and 4-hydroperoxy-2(E)-nonenal.5,6 ONE and HNE have been identified as the most abundant, toxic, and reactive lipid-derived aldehydes in biological systems.6,7 These aldehydes can modify DNA bases to produce highly mutagenic DNA adducts, and this activity is responsible for the cytotoxicity of these aldehydes.8,9 In addition, ONE and © 2019 American Chemical Society

HNE can change protein function and alter gene regulation by modifying amino acid residues and cross-linking proteins.7,10 ONE and HNE form Michael addition products with the nucleophilic amino acids Cys, His, and Lys. ONE can modify Arg and Lys through a Schiff base intermediate to produce a substituted imidazole and a stable 4-ketoamide, respectively.11,12 Angiotensin (Ang) II is the major bioactive peptide in the renin/angiotensin system (RAS) and regulates vasocontraction and cardiovascular homeostasis and remodeling.13 In addition, Ang II activates NADPH oxidase and enhances ROS production by binding to Ang II type 1 (AT1) receptor,14 which mediates the major biological effects of Ang II.13,15 The ROS then stimulate downstream kinases and mediate cardiovascular remodeling.16 Numerous studies have also demonstrated the relation between ROS induced by Ang II and cardiovascular diseases such as hypertension, atheroscleReceived: March 14, 2019 Published: May 30, 2019 1412

DOI: 10.1021/acs.chemrestox.9b00110 Chem. Res. Toxicol. 2019, 32, 1412−1422

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Chemical Research in Toxicology rosis, and heart failure.14 AT1 receptor antagonists (AT1 receptor blockers, ARBs) have been widely used for the treatment of these cardiovascular diseases.17 Another major consequence of ROS-derived damage to the cardiovascular system is lipid peroxidation, which further generates the genotoxic aldehydes ONE and HNE.18 Ang II is an octapeptide (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8) formed by the cleavage of Ang I (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7Phe8-His9-Leu10) by Ang-converting enzyme. Ang II contains amino acids that can readily react with ONE or HNE, such as the N-terminus, Asp1, Arg2, and His6, which have been identified as critical for binding to AT1 receptor and for the biological activity of Ang II.19,20 Previous studies have revealed N-terminal pyruvamide-Ang II (Ang P), Arg-modified [Arg2(ONE − H2O)]-Ang II, and the N-terminal-modified 4-ketoamide form of [N-ONE]-Ang II as the major ONEmodified Ang IIs,21,22 of which Ang P is the most abundant.22 HNE was shown to modify His6 of Ang II preferentially to form Michael addition products of [His6(HNE)]-Ang II and dehydrated Michael addition products of [His6(HNE − H2O)]-Ang II (Figure 1).22 Ang P can be formed not only

modification by lipid peroxidation and investigation of its oxidative mechanism were carried out using the metabolic labeling strategy SILFAC (stable isotope labeling by fatty acids in cell culture), in which the essential fatty acid LA is replaced by [13C18]-LA.26 Changes in the levels of 13C-labeled thiadiazabicyclo-ONE-glutathione (GSH) adduct (TOG) as well as of 13C-labeled modified Ang IIs were then measured using the SILFAC system upon treatment with Ang II in the absence or presence of ARB.

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MATERIALS AND METHODS

Materials. Human Ang II was obtained from Peptide Institute, Inc. (Osaka, Japan). ONE and HNE were purchased from Cayman chemical Co. (Ann Arbor, MI). Acetonitrile, diethyl ether, ethanol, formic acid, GSH, losartan potassium salt, Dulbecco’s phosphate buffered saline (D-PBS), and Dulbecco’s modified Eagle’s medium (DMEM) low glucose (L-glutamine, nonessential fatty acids, phenol red, sodium pyruvate) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Chelex-100 chelating ion-exchange resin (100−200 mesh size) was purchased from Bio-Rad Laboratories (Hercules, CA). Ultrapure water was obtained from a Milli-Q Integral 10 (EMD Millipore, Billerica, MA) equipped with a 0.22 μm membrane cartridge. AscA, [13C18]-LA, bovine serum albumin (BSA), and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich Inc. (St. Louis, MO). Copper(II) sulfate pentahydrate (CuSO4·5H2O) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Biological Industries Israel Beit-Haemek Ltd. (Kibbutz Beit-Haemek, Israel). Millex-GV (0.22 μm) syringe sterilization filters were obtained from Merck Millipore Ltd. (Billerica, MA). Cell Banker 3 was obtained from Nippon Zenyaku Kogyo Co. Ltd. (Fukushima, Japan). Oasis WCX and HLB solid-phase extraction (SPE) cartridges were purchased from Waters Co. (Milford, MA). Liquid Chromatography. Chromatographies for LC systems 1, 2, and 3 were carried out on an Ultimate 3000 LC system (Dionex, Sunnyvale, CA) equipped with an SRD-3600 degasser, HPG-3400 RS pump, TCC-3000SD column compartment, and WPS-3000TRS autosampler. LC systems 1 and 3 employed a SunFire C18 column (150 × 2.1 mm i.d., 3.5 μm; Waters, Milford, MA). LC system 2 employed an Accucore RP-MS column (100 × 2.1 mm i.d., 2.6 μm; Thermo Fisher Scientific Inc., Waltham, MA). For LC system 1, solvent A was water/acetonitrile (98:2, v/v) containing 0.2% (v/v) formic acid, and solvent B was acetonitrile/water (98:2, v/v) containing 0.2% (v/v) formic acid. For LC system 2, solvent A was water/methanol/acetonitrile (30:35:35, v/v/v) containing 10 mM ammonium formate, and solvent B was methanol/acetonitrile (1:1, v/ v). For LC systems 3, solvent A was water containing 0.1% (v/v) formic acid, and solvent B was acetonitrile containing 0.1% (v/v) formic acid. The linear gradient for LC system 1 was as follows: 10% B at 0 min, 35% B at 20 min, 95% B at 21 min, 95% B at 24 min, 10% B at 25 min. The linear gradient for LC system 2 was as follows: 50% B at 0 min, 85% B at 3 min, 95% B at 33 min, 50% B at 34 min. The linear gradient for LC system 3 was as follows; 3% B at 0 min, 80% B at 20 min, 95% B at 21 min, 95% B at 24 min, 3% B at 25 min. The separations using LC system 1 and 3 were performed with a flow rate of 0.2 mL/min and column oven temperature of 30 °C. The separation using LC system 2 was performed with a flow rate of 0.2 mL/min and column oven temperature of 20 °C. Mass Spectrometry. The TSQ-Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an ESI source was used in positive ion mode for LC systems 1 and 3, and in negative ion mode for LC system 2. Data were processed using Xcalibur (version 2.1.0, Thermo Fisher Scientific Inc.). Full scanning analyses were performed in the range of m/z 300−1300. Argon was used as the collision gas in collision-induced dissociation (CID) experiments coupled with tandem mass spectrometry (MS/MS) at 1.5 mTorr in the second (rf-only) quadrupole. Unit resolution was maintained for full scanning and MS/MS analyses. For the SRM

Figure 1. Modified Ang IIs identified in the reaction of Ang II with ONE/HNE.

by ONE-derived oxidative decarboxylation of the N-terminal Asp but also by hydroxyl radical-mediated reaction, which involves initial abstraction of the N-terminal α-hydrogen and hydrolysis of the ketimine intermediate.23 Subsequent investigation of the altered biological activity of modified Ang IIs revealed that the affinity of Ang P toward AT1 receptor was significantly lower than that of Ang II.24 In endothelial cell systems, Ang II is metabolized to Ang III by aminopeptidase A (APA). However, we have shown that the modified Ang II such as Ang P is not a substrate for APA.25 These results indicate that Ang II modifications by lipid hydroperoxidederived reactive aldehydes are associated with functional changes that could contribute to Ang II-induced oxidative damage of the cardiovascular system. Although the identities and biological effects of modified Ang IIs have been confirmed by in vitro experiments, their presence and functions in cell systems have not been investigated. In the present study, LC/electrospray ionization (ESI)-selected reaction monitoring (SRM)/MS was used to detect lipid hydroperoxide-derived modifications of Ang II in EA.hy926 cells subjected to oxidative stress. Ang II-induced lipid peroxidation was also examined by monitoring modified Ang IIs. Further confirmation of Ang II-induced oxidative 1413

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culture medium (500 μL) were allowed to react with sodium borohydride (0.01 M in 0.1 M NaOH, 500 μL) and extracted by SPE using Oasis WCX (1 cc, 30 mg) cartridge. Briefly, the cartridge was conditioned with acetonitrile (1 mL) and water (1 mL). The sample pH was adjusted to pH 5 using formic acid (30 μL). After the sample was loaded, the column was washed with 25 mM citrate buffer (pH 5, 1 mL) and acetonitrile (1 mL). Modified Ang IIs were eluted with water/acetonitrile (50:50, v/v) containing 5% (v/v) formic acid. Then solvent was evaporated under a stream of nitrogen. The samples were redissolved in water (100 μL). A portion of the sample (10 μL) was analyzed by LC/ESI-SRM/MS using LC system 1. Reaction of GSH with ONE. A solution of ONE (46.2 μg, 0.3 μmol) in 10 μL of methyl acetate/ethanol (1:1, v/v) was added to GSH (30.7 μg, 0.1 μmol) in the Chelex-treated 50 mM sodium phosphate buffer (pH 7.4, 190 μL). The reaction mixture was incubated at 37 °C for 24 h. After incubation, excess ONE was removed by extraction using diethyl ether (200 μL × 2). A portion of the reaction mixture was analyzed by LC/ESI-MS and MS/MS using LC system 3. Extraction of TOG. Confluent cells were washed twice with DPBS (1 mL), resuspended in water (200 μL), lysed by sonication, and then centrifuged. The supernatants were collected. A portion of sample (10 μL) was analyzed by LC/ESI-SRM/MS using LC system 3. Incubation of EA.hy926 Cells (Unlabeled/Labeled) in the Presence of Ang II, AscA, CuSO4, or Losartan. For all incubation experiments described below, EA.hy926 cells (unlabeled/labeled) were seeded at a density of 1.35 × 105 cells/well in 12-well plates in DMEM with 10% (v/v) FBS and incubated for 6 days. Cells were washed twice with D-PBS (1 mL) and subjected to the treatment in FBS-free DMEM to avoid Ang II binding to the albumin in FBS. Ang II was dissolved in 50 mM sodium phosphate buffer (pH 7.2). AscA, CuSO4, and losartan were dissolved in water. All the treating reagents were sterilized by Millex-GV (0.22 μm) syringe filter when added to the cells. Incubation of EA.hy926 Cells with Ang II in the Presence of Increasing Concentrations of AscA and CuSO4. Cells were treated with Ang II (100 μM) in the presence of AscA (0−2.0 mM) and CuSO4 (0−100 μM) and incubated at 37 °C for 24 h. After incubation, the culture medium was collected, and the modified Ang IIs were extracted by SPE as described above. Incubation of EA.hy926 Cells (Unlabeled/Labeled) with Increasing Concentrations of Ang II. Cells (unlabeled/labeled) were incubated with Ang II (0−100 μM) in the presence of AscA (1.0 mM) and CuSO4 (50 μM) at 37 °C for 24 h. After incubation, the culture medium was collected, and the modified Ang IIs were extracted by SPE, while TOG was extracted from the labeled cells as described above. Incubation of Labeled EA.hy926 Cells with Ang II and Losartan. Labeled cells were incubated with Ang II (100 μM), AscA (1.0 mM), and CuSO4 (50 μM) in the presence of losartan (0−1000 μM) at 37 °C for 24 h. After incubation, the culture medium was collected, and the modified Ang IIs were extracted by SPE as described above.

analysis, the transition, collision energy, and S-lens RF amplitude were optimized (Tables S1−S4). The operating conditions for LC systems 1 and 3 were as follows: heated capillary temperature, 220 °C; ion spray voltage, 3.0 kV; vaporizer temperature, 450 °C; sheath and auxiliary gas (nitrogen) pressures, 30 and 45 (arbitrary units), respectively. The scan width was m/z 1.00, and the scan time was 0.35 s per SRM transition. The operating conditions for LC system 2 were as follows: heated capillary temperature, 217 °C; ion spray voltage, − 4.0 kV; vaporizer temperature, 191 °C; sheath and auxiliary gas (nitrogen) pressures, 50 and 45 (arbitrary units), respectively. The scan width was m/z 1.00, and the scan time was 0.04 s per SRM transition. Cell Culture. EA.hy926 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in DMEM supplemented with 10% (v/v) FBS. The cells were grown in T-75 culture flask and maintained at 37 °C in a humidity atmosphere of 95% air/5% CO2. The medium was changed at 48−72 h intervals. For each passage, the cells were washed twice with D-PBS (5 mL) and incubated at 37 °C with 0.25% trypsin-EDTA (2 mL). The cells from passages 5−20 were used for each experiment. Preparation of [13C18]-LA-Albumin Complexes. [13C18]-LAalbumin complexes were prepared as described51 with slight modifications. In brief, [13C18]-LA (15.1 μmol) was heated at 70 °C for 1 h under a nitrogen atmosphere with 0.015 M KOH. A solution of defatted BSA (1.0 g) dissolved in the chelex-treated phosphate buffer (pH 7.2) was added to the K+ salt of [13C18]-LA, and the tube was flushed with nitrogen, capped, and placed on a shaker at 37 °C for 48 h. This gave a 3.3 mM stock solution, which was further diluted with D-PBS to give a 0.33 mM stock solution. The [13C18]-LA-albumin stock solution was membrane-filtered and then added to the culture media to give final concentrations of 4.1−16.5 μM [13C18]-LA-albumin. Stable Isotope Labeling by Fatty Acids in Cell Culture (SILFAC). Population doubling time and passage interval of EA.hy926 cells were approximately 25 h and 5−6 days, respectively. The cells in passage between 5 and 7 were used for the SILFAC experiments. EA.hy926 cells were cultured in DMEM supplemented with 10% FBS and 4.1−16.5 μM [13C18]-LA-albumin under 5% CO2 at 37 °C. Growth medium was replaced with fresh medium containing 4.1− 16.5 μM [13C18]-LA-albumin every other day (48 and 96 h) until the cells reached at about 75−85% confluency. The labeling ratio of [13C18]-LA to [12C18]-LA was checked after the incubation for 0, 2, 4, and 6 days. Cell Harvest and Lipid Extraction. Confluent cells were washed twice with D-PBS (1 mL) and scraped. The cells were then subjected to total lipid extraction by Bligh−Dyer method.27 The resulting lipids were dissolved in 3 mL of methanol/0.1% formic acid (1:9, v/v) and further purified on Oasis HLB (3 mL/60 mg) cartridges to isolate phospholipids. The cartridges were conditioned with methanol (3 mL) followed by 0.1% aqueous formic acid (3 mL). The total lipid solutions (3 mL) were loaded on the cartridges and washed with water (3 mL) and hexane (3 mL). The phospholipids were recovered by elution with methanol (3 mL) and evaporated to dryness under a stream of nitrogen. The samples were redissolved in methanol/ acetonitrile (1:1, v/v, 500 μL). A portion of the sample (10 μL) was analyzed by LC/ESI-SRM/MS using LC system 2. Reaction of Ang II with ONE/HNE. A solution of ONE (46.2 μg, 0.3 μmol) in methyl acetate/ethanol (1:1, v/v, 10 μL) and HNE (46.9 μg, 0.3 μmol) in ethanol (10 μL) was added to Ang II (209.2 μg, 0.2 μmol) in the Chelex-treated 50 mM sodium phosphate buffer (pH 7.4, 180 μL). The reaction mixture was incubated at 37 °C for 24 h. After incubation, excess ONE and HNE were removed by extraction using diethyl ether (200 μL × 2). Modified Ang IIs in the Chelex-treated 50 mM sodium phosphate buffer (pH 7.4) were allowed to react with sodium borohydride (0.1 M in 0.1 M NaOH, 200 μL) and purified by SPE described below. A portion of the solution (10 μL) was analyzed by LC/ESI-MS and MS/MS using LC system 1. Extraction of Modified Ang II. Modified Ang IIs in the Chelextreated 50 mM sodium phosphate buffer (pH 7.4) or in the cell

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RESULTS AND DISCUSSION LC/ESI-MS Analysis of ONE- and HNE-Modified Ang II. LC/ESI-MS analysis of the products from the reaction of Ang II with ONE/HNE at 37 °C for 24 h revealed the presence of Ang P ([M + 2H]2+ = m/z 501.3), [His6(HNE)]-Ang II ([M + 2H]2+ = m/z 601.8), [N-ONE]-Ang II (Michael adduct and 4ketoamide form; [M + 2H]2+ = m/z 600.8), [His6(HNE − H2O)]-Ang II ([M + 2H]2+ = m/z 592.8), and [Arg2(ONE − H2O)]-Ang II ([M + 2H]2+ = m/z 591.8), together with residual Ang II ([M + 2H]2+ = m/z 523.8), as reported previously (Figure 1).21,22 ONE- and HNE-modified Ang IIs were reacted with sodium borohydride to stabilize the Michael addition products and to improve the peak shape of Ang P. 1414

DOI: 10.1021/acs.chemrestox.9b00110 Chem. Res. Toxicol. 2019, 32, 1412−1422

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Figure 2. LC/ESI-SRM/MS analyses of the (A) reaction between Ang II (1 mM) and ONE/HNE (1.5 mM each) at 37 °C for 24 h, and (B) culture medium from EA.hy 926 cells incubated with Ang II (100 μM), AscA (1 mM), and CuSO4 (50 μM) at 37 °C for 24 h.

enhanced their production in a dose-dependent manner, and the maximum levels of Ang P and [His6(HNE)]-Ang II were detected at a concentration of 50 μM CuSO4 (Figure 3A). The level of AscA was then changed from 0 to 2 mM, while the CuSO4 level was kept at 50 μM in the presence of Ang II (100 μM). An AscA dose-dependent elevation in the formation of Ang P and [His6(HNE)]-Ang II was observed (Figure 3B). These results indicate that Ang II modifications likely occur not only in vitro but also in vivo, and their levels could reflect the degree of oxidative stress. Further investigation may be required in other endothelial cells or the primary cell culture because EA.hy926 cells used in the current study is a hybrid cell line, although it is reported to maintain many of endothelial cell functions.28,29 Effect of Increasing Concentrations of Ang II on Modified Ang IIs. Ang II was shown to increase oxidative stress in a dose- and time-dependent manner in the cell system.30 Our results also support Ang II-induced oxidative stress. Thus, EA.hy926 cells were incubated with Ang II (0− 100 μM), AscA (1 mM), and CuSO4 (50 μM), and the modified Ang IIs were analyzed as described above. The levels of modified Ang IIs formed in the incubation with 1 μM Ang II were close to the detection limit of our current LC/ESI-SRM/ MS conditions. Therefore, up to 100 μM of Ang II was used to observe its dose-dependent effect clearly. In the absence of Ang II, all modified Ang IIs monitored were under the detection limit. Ang P and [His6(HNE)]-Ang II were detected at 1 μM (Figure 4A) and 10 μM Ang II (Figure 4B), respectively. The levels of both adducts then increased in a dose-dependent manner (Figure 4C). Elevated levels of circulating and intrarenal Ang II have been associated with various cardiovascular diseases.31−33 The increased production of ROS induced by Ang II causes lipid peroxidation with the

Upon reduction, the N-terminal carbonyl carbon of Ang P becomes a chiral center that leads to separation of the two epimers. The retention times of Ang II and modified Ang IIs on LC system 1 were as follows: Ang II, 5.5 min; Ang P ([M + 2H]2+ = m/z 502.3, + 2H), 8.4 and 8.5 min; [His6(HNE)]Ang II ([M + 2H]2+ = m/z 602.9, + 2H), 10.6 min; [N-ONE]Ang II (Michael addition product; [M + 2H]2+ = m/z 602.9, + 4H), 10.6 min; [N-ONE]-Ang II (4-ketoamide form) ([M + 2H]2+ = m/z 601.9, + 2H), 13.8 and 13.9 min; [His6(HNE − H2O)]-Ang II, 13.7−14.0 min; [Arg2(ONE − H2O)]-Ang II ([M + 2H]2+ = m/z 591.83, + 2H), 15.0−15.5 min (Figure S1). To analyze the modified Ang IIs in the cell system, SRM transitions were determined on the basis of their MS and MS/ MS spectra (Figure S2). [M + 2H]2+ ion was set as Q1, and the most intense product ion, y2 ion (m/z 263.1) or HNEmodified His (m/z 268.1), was set as Q3. The collision energy (CE) was changed from 5 to 80 eV; the CE giving the maximum intensity was chosen for each transition. S-Lens RF amplitude was also optimized for each transition (Table S1). LC/ESI-SRM/MS analysis of the reaction mixture of Ang II and ONE/HNE after reduction and SPE cleanup is shown in Figure 2A. Formation of Modified Ang IIs in EA.hy 926 Cells Subjected to Oxidative Stress. EA.hy926 cells were incubated with Ang II (100 μM), AscA (1 mM), and CuSO4 (50 μM) at 37 °C for 24 h. Modified Ang IIs in the medium were then analyzed using the optimized LC/ESI-SRM/MS method after reduction and SPE. Ang P and [His6(HNE)]-Ang II were detected at retention times of 8.2/8.3 and 10.5 min, respectively (Figure 2B). The presence of both adducts was also observed when the cells were incubated with Ang II (100 μM) and AscA (1 mM). The addition of CuSO4 (0−100 μM) 1415

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Figure 3. Formation of Ang P (gray bars) and [His6(HNE)]-Ang II (white bars) in EA.hy 926 cells incubated with Ang II (100 μM) in the presence of (A) AscA (1 mM) and CuSO4 (0−100 μM), (B) AscA (0−2 mM), and CuSO4 (50 μM) at 37 °C for 24 h. (Data are from a single experiment. In the preliminary experiments, concentration range (CuSO4 and AscA) and incubation time were optimized and dose-dependent increases in the formation of Ang P and [His6(HNE)]-Ang II were confirmed.)

dependent MS is used to analyze peptides modified by 1:1 ratios of the 12C and 13C aldehyde isomers. Using this methodology, the major lipid hydroperoxide-mediated protein modifications in MCF-7 cells were identified without the need for chemical labeling or further affinity purification. In the present study, SILFAC was modified to allow for the full incorporation of [13C18]-LA to confirm Ang II-induced oxidative modification via lipid peroxidation in EA.hy926 cells. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were analyzed by LC/ESI-SRM/MS in negative ion mode to ensure the incorporation of [13C18]-LA in EA.hy926 cells. PC and PE were detected as [M + HCO2]− and [M − H]− ions, respectively. CID can easily fragment acyl moieties at the sn-1 and -2 positions of PC and PE. For example, MS analysis of 1-palmytoyl-2-linoleoyl-sn-glycero-3-PC (PLPC) exhibited [M + HCO2]− at m/z 802.2. The MS/MS spectrum showed product ions at m/z 255.2 and m/z 279.2, which correspond to sn-1 and -2 fragmentations, respectively. MS analysis of PLPE exhibited [M − H]− at m/z 714.4, which produced ions at m/z 255.0 (sn-1) and m/z 279.0 (sn-2) by MS/MS analysis (Figure S3). The MS and MS/MS spectra of 1-stearoyl-2-linoleoyl-sn-glycero-3-PC (SLPC) and SLPE (Figure S4) showed similar patterns to those of PLPC and PLPE. On the basis of these results, SRM transitions were determined (Table S2). S-Lens RF amplitude and CE were optimized using standard PCs (C16:0/C18:2, C18:0/18:2) and PEs (C16:0/C18:2, C18:0/C18:2). For the analysis of

generation of genotoxic ONE and HNE, which readily modify Ang II as well as other cellular molecules. The formation of ONE- and HNE-modified Ang II could alter cardiovascular function by modulating the biological activity of Ang II. Optimization of Conditions for SILFAC. Stable isotope labeling techniques have been applied to LC−MS-based biomarker discovery and the relative/absolute quantification of proteins/peptides.34,35 The introduction of stable isotopes into proteins/peptides is accomplished by either tagging with chemical reagents or metabolic labeling of cells in culture. Representative chemical tagging methods include isotopecoded affinity tags (ICAT)36 and isobaric tags for relative and absolute quantitation (iTRAQ).37 In stable isotope labeling by amino acids in cell culture (SILAC),38 cells are grown in medium containing labeled amino acids. A stable isotopelabeled proteome (SILAP) has been used as an internal standard (IS).34,39 In addition, the absolute quantitation of target peptides or proteins was previously accomplished by using synthetic isotope-labeled peptides (AQUA peptides)35 or Edman degradation reaction with [13C6]-phenylthiohydantoinamino acid.40,41 The metabolic labeling strategy SILFAC was recently developed to screen protein modifications resulting from lipid peroxidation.26 The stable isotope [13C]-labeled essential fatty acid LA is added to a cell culture and allowed to incorporate into the lipidome until the LA to [13C18]-LA ratio is 1:1. Under oxidative stress conditions, cellular LA and [13C18]-LA can decompose to aldehydes, which react with proteins. After protein extraction and digestion, isotope pattern 1416

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Figure 4. LC/ESI-SRM/MS analyses of (A) Ang P and (B) [His6(HNE)]-Ang II in EA.hy926 cells incubated with Ang II (0−100 μM) in the presence of AscA (1 mM) and CuSO4 (50 μM) at 37 °C for 24 h. (C) Formation of Ang P (black bars) and [His6(HNE)]-Ang II (white bars) presented as means ± SEM for three independent experiments.

[13C18]-phospholipid) × 100, which was shown to increase in a [13C18]-LA dose-dependent manner (Figure S6). The metabolic incorporation of [13C18]-LA was highest when the cells were treated with 16.5 μM [13C18]-LA, and thus, this condition was employed for SILFAC. We noted that cell viability significantly decreased at [13C18]-LA culture medium concentrations above 16.5 μM (data not shown). Formation of Modified Ang IIs in SILFAC-Treated Labeled EA.hy 926 Cells Incubated with Ang II. We used the LC/ESI-SRM/MS method optimized above to analyze the modified Ang IIs in the labeled EA.hy926 cells. In SRM transitions for Ang IIs modified by labeled aldehydes, m/z values shifted by + 4.5 Da were set as Q1, except for Ang P. The pyruvamide moiety of ONE-derived Ang P is formed

phospholipids containing [13C18]-LA, values shifted by + 18 Da were set as Q1 and Q3 (sn-2 fragmentation) ions (Table S2). Using these optimized methods, the concentration and incubation time of [13C18]-LA-albumin were optimized to achieve SILFAC in EA.hy926 cells. EA.hy926 cells were incubated for 6 days, with three rounds of [13C18]-LA (4.1− 16.5 μM) treatment at 0, 48, and 96 h. After incubation for 6 days, phospholipids were extracted from the cells and subjected to SPE cleanup. LC/ESI-SRM/MS analysis of the extracted phospholipids revealed metabolic incorporation of [13C18]-LA (Figure S5). The metabolic incorporation of [13C18]-LA into phospholipid was estimated using the equation as follows: MS peak intensity of [13C18]-phospholipid/(MS peak intensity of [12C18]-phospholipid + MS peak intensity of 1417

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Figure 5. LC/ESI-SRM/MS analyses of (A) Ang P and (B) [His6([13C9]-HNE)]-Ang II in labeled EA.hy926 cells incubated with Ang II (0−100 μM) in the presence of AscA (1 mM) and CuSO4 (50 μM) at 37 °C for 24 h. (C) Formation of Ang P (black bars) and [His6([13C9]-HNE)]-Ang II (white bars) presented as means ± SEM for three independent experiments.

Ang P (Figure 5A) and [His6([13C9]-HNE)]-Ang II (Figure 5B) in the cell system. Furthermore, their MS peak areas increased in an Ang II dose-dependent manner, as shown in Figure 5C. Formation of [13C9]-TOG in SILFAC-Treated Labeled EA.hy 926 Cells Incubated with Ang II. Lipid hydroperoxide-derived modifications can occur in various cellular biomolecules including Ang II. It was reported that ONE readily reacts with GSH to produce the stable adduct TOG (Figure S7).42 We investigated Ang II-induced lipid peroxidation-derived modifications of biomolecules other than Ang

through the hydrolysis of an aldehyde Schiff base intermediate.21,22 Thus, Ang Ps produced by unlabeled and labeled ONE should have identical [M + 2H]2+ values for Q1. In addition, y2 ion at m/z 263.1 was not modified by ONE or HNE. The Q3 ion for [His6([13C9]-HNE)]-Ang II was shifted by + 9 Da because [13C9]-HNE-modified His was selected as a product ion in the SRM transition (Table S3). The labeled cells were incubated with Ang II (0−100 μM) in the presence of AscA (1 mM) and CuSO4 (50 μM), and the modified Ang IIs in the cell culture medium were analyzed after reduction and SPE. LC/ESI-SRM/MS analyses revealed the presence of 1418

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Figure 6. LC/ESI-SRM/MS analyses of (A) [13C9]-TOG in labeled EA.hy926 cells incubated with Ang II (0−100 μM) in the presence of AscA (1 mM) and CuSO4 (50 μM) at 37 °C for 24 h. (B) Formation of TOG presented as means ± SEM for three independent experiments.

II by monitoring labeled TOG in SILFAC-treated labeled EA.hy926 cells incubated with Ang II. Prior to the cell experiments, LC/ESI-SRM/MS conditions for the analysis of TOG were optimized using the reaction mixture of authentic GSH and ONE. For the absolute quantification, IS could be prepared using a labeled GSH. The retention time of TOG on LC system 3 was 13.4 min (Figure S8A). MS/MS analysis of [M + H]+ (m/z 426.1) resulted in the formation of a major product ion at m/z 279.9, which corresponded to the loss of CONHCH2CO2H-CONH2 (Figure S8B). The SRM transitions for TOG and [13C9]-TOG (modified by [13C9]-ONE) were then set as m/z 426.1 → m/z 279.9 and m/z 435.1 → m/ z 288.9, respectively (Table S4). After the labeled cells were incubated with Ang II (0−100 μM) in the presence of AscA (1 mM) and CuSO4 (50 μM), the cells were washed and collected. [13C9]-TOG was extracted from the cells by sonication in water and analyzed. LC/ESI-SRM/MS analyses revealed the presence of [13C9]-TOG even without Ang II treatment (Figure 6A), which indicated the basal level of oxidative stress. The concentration of [13C9]-TOG then increased in an Ang II dose-dependent manner (Figure 6B). These results support the findings of the current study obtained using unlabeled EA.hy926 cells, namely that Ang II can induce oxidative stress and lipid peroxidation, followed by the production of ONE and HNE, which modify both Ang II and other biological molecules. Effect of ARB on Ang II-Induced Oxidative Stress. Ang II stimulates AT1 receptor, resulting in activation of NADPH oxidase and phospholipase A2, which enhance the production of ROS and the release of LA and AA, respectively.14,43,44 AT1

receptor mediates most of the known physiological effects of Ang II on the cardiovascular system.13,15 Thus, Ang II-induced lipid peroxidation-derived modifications could be mediated by AT1 receptor. ARBs exert antioxidative effects45,46 and have been widely used for the treatment of hypertension and other cardiovascular conditions.17,47 The effect of ARBs on lipid hydroperoxide-derived modifications was investigated using the SILFAC-treated cell system. Labeled EA.hy926 cells were incubated with Ang II (100 μM), AscA (1 mM), and CuSO4(50 μM) in the presence of losartan (0−1000 μM), a well-known ARB. In the absence of losartan, Ang P and [His6([13C9]-HNE)]-Ang II were detected, as shown in the first SRM/MS chromatograms in Figure 7A and B, respectively. In contrast, the formation of Ang P and [His6([13C9]-HNE)]-Ang II was dramatically inhibited by losartan in a dose-dependent manner (Figure 7C). In separate cell-free reactions of Ang II (100 μM) with ONE/HNE (150 μM each), AscA (1 mM)/CuSO4(50 μM), or ONE/HNE (150 μM each)/AscA (1 mM)/CuSO4(50 μM), the addition of losartan (1000 μM) did not affect the levels of modified Ang II produced (data not shown). Clinical studies have previously demonstrated that losartan decreases oxidative stress and suppresses inflammation.46,48 The results obtained in the present study indicate that lipid hydroperoxide-derived modifications are at least partially due to the activation of the AT1 receptor and that losartan may protect the cardiovascular system by inhibiting lipid hydroperoxidederived damage. 1419

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Figure 7. LC/ESI-SRM/MS analyses of (A) Ang P and (B) [His6([13C9]-HNE)]-Ang II in labeled EA.hy926 cells incubated with Ang II (100 μM), AscA (1 mM), and CuSO4 (50 μM) in the presence of losartan (0−1000 μM) at 37 °C for 24 h. (C) Formation of Ang P (black bars) and [His6([13C9]-HNE)]-Ang II (white bars) presented as means ± SEM for three independent experiments.

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CONCLUSIONS

the production of modified Ang IIs in a dose-dependent manner. Ang II is synthesized not only in the circulatory system but also in various organs such as the heart, brain, kidneys, and blood vessel walls.13 The presence of a functional local RAS in pancreas and adipose tissue was also reported.49 Increased production of Ang II has been observed in cells cultured in high glucose and in conscious diabetic rats, indicating an important link between dysregulated RAS and type 2 diabetes as well as cardiovascular disorders and hypertension.49−51 In addition to Ang II, other Ang peptides, including Ang (1−7), Ang III, Ang IV, and Ang A, have been

This study demonstrated that Ang II induces lipid peroxidation by activating the AT1 receptor. The lipid hydroperoxides formed were decomposed to the reactive aldehydes ONE and HNE, which modified cellular Ang II and other molecules such as GSH. Modifications to endogenous Ang II and GSH were unequivocally characterized in cells labeled metabolically with [13C18]-LA. Modified Ang II-[13C9] and GSH adduct-[13C9] were detected, and their levels were elevated in an Ang II dosedependent manner. Finally, the involvement of the AT1 receptor was confirmed using ARB losartan, which inhibited 1420

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Chemical Research in Toxicology shown to be involved in vascular regulation.52 A previous study demonstrated that these Ang peptides readily react with ONE, HNE, and ROS to produce modifications at the N-terminus, Arg, and His, which are similar to those on Ang II.23 Our current studies are focused on detecting modified Ang peptides and examining their roles in biological systems.

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standard; LA, linoleic acid; LOX, lipoxygenase; MS/MS, tandem mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; m/z, mass-to-charge; ONE, 4-oxo-2(E)-nonenal; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLPC, 1-palmytoyl-2-linoleoyl-snglycero-3-phosphatidylcholine; PLPE, 1-palmytoyl-2-linoleoylsn-glycero-3-phosphatidylethanolamine; PUFA, polyunsaturated fatty acid; RAS, renin/angiotensin system; ROS, reactive oxygen species; SILFAC, stable isotope labeling by fatty acids in cell culture; SLPC, 1-stearoyl-2-linoleoyl-sn-glycero-3phosphatidylcholine; SLPE, 1-stearoyl-2-linoleoyl-sn-glycero3-phosphatidylethanolamine; SPE, solid-phase extraction; SRM, selected reaction monitoring; TOG, thiadiazabicyclo-4oxo-2(E)-nonenal-glutathione adduct

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.9b00110. SRM conditions for analysis of Ang II and modified Ang IIs, [12C18]- and [13C18]-phospholipids, Ang II and modified Ang IIs in labeled EA.hy 926 cells, and TOG and [13C9]-TOG; LC/ESI-MS and MS/MS analyses of Ang II modified by ONE and HNE; MS and MS/MS analyses of PLPC, PLPE, SLPC, and SLPE; LC/ESISRM/MS analyses of [12C18]- and [13C18]-phospholipids, and incorporation of [13C18]-LA by SILFAC into phospholipids in EA.hy926 cells; proposed mechanism, and LC/ESI-MS and -MS/MS analyses of TOG (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +81-22-795-6818. *E-mail: [email protected]. ORCID

Seon Hwa Lee: 0000-0003-1095-5516 Tomoyuki Oe: 0000-0002-3893-0815 Present Addresses

‡ S.F., Pharmaceutical Technology Division, Daiichi Sankyo Co. Ltd., 1−12−1 Shinomiya, Hiratsuka, Kanagawa 254−0014, Japan. § R.T., Kobe Pharmaceutical Research Center, Nippon Boehringer Ingelheim Co. Ltd., 6−7−5 minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 660−0047, Japan.

Funding

This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research (to T.O., 15K14935 for 2015−2016), and Grants-in-Aid for Scientific Research (C) (to S.H.L., 16K08391 for 2016−2018) and (B) (to T.O., 16H05078 for 2016−2018) from the Japan Society for the Promotion of Science (JSPS). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Technical Support Center at Tohoku University for the use of TSQ-Vantage. ABBREVIATIONS AA, arachidonic acid; Ang, angiotensin; Ang P, pyruvamideAng II; APA, aminopeptidase A; ARB, angiotensin II type 1 receptor blocker; AscA, L-ascorbic acid; AT1, angiotensin II type 1; BSA, bovine serum albumin; CE, collision energy; CID, collision-induced dissociation; COX, cyclooxygenase; DMEM, Dulbecco’s modified Eagle’s medium; D-PBS, Dulbecco’s phosphate buffered saline; EDTA, ethylenediaminetetraacetic acid; ESI, electrospray ionization; FBS, fetal bovine serum; GSH, glutathione; HNE, 4-hydroxy-2(E)-nonenal; IS, internal 1421

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