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Chem. Res. Toxicol. 2007, 20, 1170–1176
Upregulation of Cyclooxygenase-2 by Motorcycle Exhaust Particulate-Induced Reactive Oxygen Species Enhances Rat Vascular Smooth Muscle Cell Proliferation Huei-Ping Tzeng,†,‡ Rong Sen Yang,‡,§ Tzuu-Huei Ueng,† and Shing-Hwa Liu*,†,| Institute of Toxicology and Department of Orthopaedics, College of Medicine, National Taiwan UniVersity, Taipei, Taiwan, and Department of Surgery and Department of Emergency Medicine, National Taiwan UniVersity Hospital, Taipei, Taiwan ReceiVed March 19, 2007
Long-term exposure to particulate air pollution has been implicated as a risk factor for cardiovascular disease and mortality. Short-term exposure has also been suggested to contribute to complications of atherosclerosis. Aberrant regulation of smooth muscle cell proliferation is thought to associate with the pathophysiology of vascular disorders such as atherosclerosis. In this study, we investigate the influence of organic extracts of motorcycle exhaust particulates (MEPE) on rat vascular smooth muscle cell (VSMC) proliferation and related regulation signaling. Exposure of VSMCs to MEPE (10–100 µg/mL) enhanced serum-induced VSMC proliferation. The expression of proliferating cell nuclear antigen (PCNA) was also enhanced in the presence of MEPE. VSMCs treated with MEPE induced the increase in the extent of cyclooxygenase (COX)-2 mRNA and protein expression and prostaglandin E2 production, whereas the level of COX-1 protein was unchanged. Moreover, MEPE increased the production of reactive oxygen species (ROS) in VSMCs in a dose-dependent manner. MEPE could also trigger time-dependently extracellular signal-regulated kinase (ERK)1/2 phosphorylation in VSMCs, which was attenuated by antioxidants N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate (PDTC). The level of translocation of nuclear factor (NF)-κB-p65 in the nuclei of VSMCs was also increased under MEPE exposure. The potentiating effect of MEPE on serum-induced VSMC proliferation could be abolished by COX-2 selective inhibitor NS-398, specific ERK inhibitor PD98059, and antioxidants NAC and PDTC. Taken together, these findings suggest that MEPE may contribute to the enhancement of the pathogenesis of cardiovascular diseases by augmenting proliferation of VSMCs through a ROS-regulated ERK1/2-activated COX-2 signaling pathway. Introduction Recent evidence suggests that exposure to particulate air pollution contributes to cardiovascular morbidity and mortality (1–3). A recent epidemiologic study has produced evidence of an association between atherosclerosis and ambient air pollution (4). A case-crossover study by Pope et al. has also demonstrated that short-term particulate exposures contributed to acute coronary events, especially among patients with underlying coronary artery disease (2). Moreover, several studies have shown that diesel exhaust or diesel exhaust particles or its extracts have been shown to affect the cardiovascular system (5–7). An epidemiological study in eight European cities has indicated that particulate air pollution has a significant effect on admissions for cardiac causes and suggested that the primary effect was likely to be mainly attributable to diesel exhaust (8). The motorcycle is one of the important transportation modes in many modern cities. In Taiwan, there are more than 12 million registered motorcycles, which is the highest motorcycle per * To whom correspondence should be addressed. Telephone: (+886)2-23123456, ext. 8605. Fax: (+886)-2-23410217. E-mail:
[email protected]. † Institute of Toxicology, College of Medicine, National Taiwan University. ‡ These authors contributed equally to this work. § Department of Orthopaedics, College of Medicine, National Taiwan University. | National Taiwan University Hospital.
capita density (two persons per motorcycle) in the world (9). In cities where motorcycles are a popular means of transportation, the motorcycle exhaust (ME) emissions are a major source of air pollution. The two- and four-stroke motorcycle engines have smaller capacities and poorer combustion efficiencies than diesel and gasoline engines. The exhaust released by motorcycle combustion contains hundreds of organic compounds, including plenty of polycyclic aromatic hydrocarbons (PAHs) (10–12), similar to those reported from diesel engine emission. Motorcycle exhaust particulates (MEP) or MEP extracts (MEPE) 1 have been shown to be mutagenic (13) and genotoxic in vitro (14), neurotoxic (15) and immunotoxic in vivo (16), and antiestrogenic in vitro and in vivo (17). It has also been shown that MEPE was capable of impairing endothelium-dependent vasorelaxation in rat aorta (18) and enhancing vasoconstriction elicited by phenylephrine in the organ cultures of both intact and endothelium-denuded rat aortas (19). Vasoconstriction and proliferation of vascular smooth muscle cells (VSMCs) are two of features of atherosclerosis. However, little is known about the pathophysiological effects of the MEP on VSMCs. Reactive oxygen species (ROS) have been known to induce VSMC growth and endothelial cell damage and cardiac remod1 Abbreviations: MEPE, organic extract of motorcycle exhaust particulates; VSMCs, vascular smooth muscle cells; ROS, reactive oxygen species; NAC, N-acetylcysteine; PDTC, pyrrolidinedithiocarbamate; MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; COX-2, cyclooxygenase-2.
10.1021/tx700084z CCC: $37.00 2007 American Chemical Society Published on Web 07/24/2007
Motorcycle Exhaust and Vascular Cell Proliferation
eling, which were associated with the pathogenesis of atherosclerosis and several other cardiovascular diseases (20, 21). Our recent study has shown that MEPE enhanced vasoconstriction via a ROS-related Ca2+/myosin light chain kinase-dependent pathway (19). Moreover, mitogen-activated protein kinases (MAPKs) have been shown to be sensitive to oxidative stress (22, 23). MAPKs play an important role in cell differentiation, growth, apoptosis, and the regulation of a variety of transcription factors and gene expressions (24). H2O2 has also been demonstrated to stimulate extracellular signal-regulated kinase (ERK)/ MAPK-mediated cyclooxygenase (COX)-2 expression and prostaglandin (PG) formation in cardiomyocytes (25). In a number of cell and animal models, induction of COX-2 has been shown to promote cell growth, inhibit apoptosis, and enhance cell motility and adhesion (26). COX-2 has also been found to be expressed in atherosclerotic lesions (27). With this in mind, we performed this study to investigate the effect of MEPE on VSMC proliferation, to examine whether the ROSrelated pathway is involved, and to determine the roles of ERK and COX-2 in the MEPE-induced responses.
Materials and Methods Preparations of the Organic Extract of Motorcycle Exhaust Particulates. Preparation of MEPE was according to the method previously described (12, 19). MEPE were collected from the 0.5 µm quartz fiber filters of a Yamaha motorcycle with a 50 cm3 two-stroke engine using 95% octane unleaded gasoline. The engine was running at 150 rpm on an empty load, and the pump was set at a flow rate of 20 L/min to collect the motorcycle exhaust particulates for 30 min twice daily. It has been determined that the original concentrations of ME particles (mean values) were 118 mg/m3 for PM1, 216 mg/m3 for PM2.5, and 228 mg/m3 for PM10 (12). The extraction of motorcycle exhaust particulates was carried out in the dark for 24 h using a Soxhlet extraction method [solvent system, dichloromethane and hexane (1:1)]. The crude MEPE was vacuum-evaporated to dryness and kept desiccated at -20 °C. Ueng et al. (12) have found that qualitative GC–MS analysis of MEPE identified the presence of 14 PAHs in the chemical mixture. Carcinogenic benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-c,d)pyrene, and benzo(g,h,i)perylene and noncarcinogenic PAH such as naphthalene and phenanthrene were identified in the MEPE. Moreover, Ueng et al. (12) have described that the yield of MEPE from MEP was 56% (g/g); consequently, 100 µg/mL MEPE was equivalent to 179 µg/mL (1.79 × 105 mg/m3) MEP in cell culture medium. Primary Vascular Smooth Muscle Cell Culture. Vascular smooth muscle cells (VSMCs) were obtained from the thoracic aortas of Wistar rats according to the method previously described (19). In brief, male Wistar rats were purchased from the Animal Center of the College of Medicine, National Taiwan University. The study was conducted in accordance with the guidelines of the Animal Research Committee of the College of Medicine, National Taiwan University, for the care and use of laboratory animals. Rats (200–250 g) were sacrificed, and the thoracic aortas were removed, cleaned of fat and adventitia, cut into small strips, and then incubated in 1 mg/mL collagenase and 0.125 mg/mL elastase at 37 °C for 60 min. The cells were seeded into 10 mm diameter dishes and maintained in 0.5 mL of DMEM containing 10% fetal bovine serum at 37 °C. The cells that exhibit characteristics of VSMCs were used between the third and sixth passages. Cells were grown to 60–80% confluence, at which time they were rendered quiescent by the DMEM containing 0.1% bovine serum albumin (BSA) and maintained for 48 h before experimentation. MEPE were dissolved in dimethylsulfonyl oxide (DMSO) and added to the medium. The concentration of DMSO in the medium was less than 0.1%. Cell Proliferation Assay. 1. MTS Assay. Cell proliferation was assessed using a nonradioactive cell proliferation assay kit (CellTiter
Chem. Res. Toxicol., Vol. 20, No. 8, 2007 1171 96 Aqueous, Promega). Briefly, cells were seeded into 96-well plates overnight and starved for 48 h. Then, the medium was aspirated, and cells were cultured in DMEM containing 2% fetal bovine serum with DMSO only or various concentrations of MEPE solubilized in DMSO for 48 h. In some experiments, cells were pretreated with PD98059 (a specific ERK inhibitor), N-acetylcysteine (NAC, antioxidant), or pyrrolidinedithiocarbamate (PDTC, antioxidant) for 30 min following the treatment of MEPE for 48 h. Subsequently, the medium was removed, and cells were incubated with a mixture of tetrazolium compound [3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)] in medium for 1 h at 37 °C, which would be metabolized to a soluble formazan, and then measured in an ELISA reader at 490 nm. 2. Analysis of Cell Number. Cells were seeded into 12-well plates and allowed to attach overnight. Cells were growth arrested for 48 h and then challenged with or without MEPE in DMEM containing 2% fetal bovine serum for 3 days. Cells were harvested, and a 50 µL aliquot was mixed with 0.04% trypan blue; cells were counted twice on a hemocytometer. Detection of Intracellular ROS. Intracellular ROS generation was monitored by flow cytometry using a peroxide-sensitive fluorescent probe [2′,7′-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes)] as previously described (19). In brief, experiments were performed under dim light. Subconfluent and serumdeprived VSMCs were loaded with 10 µM DCFH-DA for 30 min after the treatment of MEPE, then chilled on ice, and washed with cold PBS. Washed cells were detached from culture plates by trypsin digestion. The fluorescence intensities for samples of 10 000 cells each were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Western Blot Analysis. Equal amounts of proteins (30 µg per lane) were subjected to 8 to 10% SDS–PAGE and transferred to nitrocellulose membranes (Amersham). The membranes were blocked with 5% fat-free milk or 3% BSA in PBST for 1 h and followed by immunoblotting with primary antibodies. Subsequently, membranes were washed three times with PBST and incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies, again followed by three washes. The signals were then visualized with an enhanced chemiluminescence detection system. Exposures were recorded on X-film. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Total cellular RNA was extracted from VSMCs by using Trizol (GIBCO), and cDNA was reverse-transcribed from 5 µg of total cellular RNA using random hexamer primers (Promaga) and murine leukemia virus reverse transcriptase (GIBCO). Two micrograms of cDNA was amplified for 30 cycles using the following rat COX-2 gene-specific primers: 5′-ACTTGCCTCACTTTGTTGAGTCATTC-3′ (sense) and 5′-TTTGATTAGTACTGTAGGGTTAATG-3′ (antisense). The cycling parameters were as follows: 1 min at 94 °C for denaturation, 1 min at 57 °C for primer annealing, and 1 min at 72 °C for polymerization. Meanwhile, the same amount of cDNA was amplified for 20 cycles using specific β-actin primers: 5′-TTGTAACCAACTGGGACGATATGG-3′(sense)and5′-GATCTTGATCTTCATGGTGCTAGG3′ (antisense). The PCR products were electrophoresed through a 1.2% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining. Preparation of Nuclear Extracts. Cells seeded in 10 cm plates were serum-deprived in DMEM containing 0.1% bovine serum albumin (BSA) for 24–48 h and treated as indicated. Cells were harvested and spun down for 5 min at 500g and 4 °C. Cells were aspirated with PBS, resuspended in 250 µL of ice-cold hypotonic buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and 0.2 mM PMSF], and incubated for 15 min on ice. The nuclei were pelleted by centrifugation at 10 000 rpm for 2 min at 4 °C. The supernatant is a cytoplasmic fraction. Nuclear pellets were washed with 1 mL of hypotonic buffer, and nuclei were spun down for 5 min at 10000g and 4 °C. The nuclear pellets were resuspended in 80 µL of ice-cold hypertonic buffer [20 mM
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Figure 1. MEPE-enhanced serum-induced VSMC proliferation. Subconfluent, growth-arrested cells were cultured in DMEM containing 2% fetal bovine serum in the absence (control, CTL) or presence of MEPE (10–100 µg/mL) for 12–72 h [48 (A), 72 (B), or 12–24 h (C)]. Cell proliferation (A), cell number (B), and proliferating cell nuclear antigen (PCNA) (C) expression were assessed with a MTS assay, a trypan blue assay, and Western blotting, respectively, as described in Materials and Methods. Data are presented as means ( sem from three independent experiments performed in duplicate. Asterisks denote P values of
