Research article

Received: 17 December 2014, Revised: 4 February 2015, Accepted: 4 February 2015 Published online in Wiley Online Library: 15 April 2015

(wileyonlinelibrary.com) DOI 10.1002/jat.3143

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PM2.5-induced oxidative stress increasesadhesion molecules expression in humanendothelial cells through the ERK/AKT/NF-κB-dependent pathwayWei Ruia, Longfei Guana, Fang Zhanga*, Wei Zhang and Wenjun Dinga*

ABSTRACT: The aim of this study was to explore the intracellular mechanisms underlying the cardiovascular toxicity of air partic-ulate matter (PM) with an aerodynamic diameter of less than 2.5μm (PM2.5) in a human umbilical vein cell line, EA.hy926. Wefound that PM2.5 exposure triggered reactive oxygen species (ROS) generation, resulting in a significant decrease in cell viability.Data fromWestern blots showed that PM2.5 induced phosphorylation of Jun N-terminal kinase ( JNK), extracellular signal regula-tory kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), and activation of nuclear factorkappa B (NF-κB). We further observed a significant increase in expressions of intercellular adhesion molecule-1 (ICAM-1) andvascular adhesionmolecule-1 (VCAM-1) in a time- and dose-dependentmanner.Moreover, the adhesion ofmonocytic THP-1 cellsto EA.hy926 cells was greatly enhanced in the presence of PM2.5. However, N-acetylcysteine (NAC), a scavenger of ROS, preventedthe increase of ROS generation, attenuated the phosphorylation of the above kinases, and decreased the NF-κB activation aswellas the expression of ICAM-1 and VCAM-1. Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and NF-κB inhibitor(BAY11-7082) significantly down-regulated PM2.5-induced ICAM-1 and VCAM-1 expression as well as adhesion of THP-1 cells,but not JNK inhibitor (SP600125) and p38MAPK inhibitor (SB203580), indicating that ERK/AKT/NF-κB is involved in the signalingpathway that leads to PM2.5-induced ICAM-1 and VCAM-1 expression. These findings suggest PM2.5-induced ROSmay function assignalingmolecules triggering ICAM-1 and VCAM-1 expressions through activating the ERK/AKT/NF-κB-dependent pathway, andfurther promoting monocyte adhesion to endothelial cells. Copyright © 2015 John Wiley & Sons, Ltd.

Keywords: PM2.5; oxidative stress; adhesion factor; ERK/AKT/NF-κB; reactive oxygen species; EA.hy926 cells

*Correspondence to: Fang Zhang and Wenjun Ding, Laboratory of Environmentand Health, College of Life Sciences, University of Chinese Academy of Sciences.No. 19A Yuquan Road, Beijing 100049, China.E-mail: dingwj@ucas.ac.cn; zhangfang@ucas.ac.cn

aLaboratory of Environment and Health, College of Life Sciences, University ofChinese Academy of Sciences, No. 19A Yuquan Road, Beijing100049, China

IntroductionAirborne fine particles with a mean aerodynamic diameter of lessthan 2.5μm (PM2.5), a component of air pollution, has been epide-miologically associated with cardiovascular diseases (Peters et al.,1997; Pope et al., 2004; Alfaro-Moreno et al., 2007; Laden et al.,2006; Riediker, 2007). Accumulating evidences in vivo studies havebeen proposed that exposure to environmentally relevant inhaledparticulate matter enhances atherosclerosis (AS) through induc-tion of vascular reactive oxygen species (ROS) (Ying et al., 2009;Hemmingsen et al., 2011; Wauters et al., 2013). In the cardiovascu-lar system, expression of cell adhesion molecules is one of the ini-tial events by activated endothelium. Endothelial activation,characterized by increased inflammation, is an early event in endo-thelial dysfunction (Horstman et al., 2004). Cell adhesionmoleculesinduce monocytes adhesion and transmigration to thesubendothelial space (Wood et al., 1993; Krieglstein and Granger,2001). Normally, intercellular adhesion molecule-1 (ICAM-1,CD54) and vascular adhesion molecule-1 (VCAM-1, CD106), as im-portant cell adhesion molecules, are members of the immuno-globulin superfamily of proteins and crucially mediates theadhesion of lymphocytes, monocytes, eosinophils and basophilsto vascular endothelium (Hubbard and Rothlein, 2000; Barreiroet al., 2002; Yang et al., 2005). The interaction between ICAM-1and VCAM-1 and their specific ligands expressed onmicrovascularendothedlial cells could be involved in cell adhesion (Collins et al.,2000; Cybulsky et al., 2001). ICAM-1 and VCAM-1 expressions are

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the consequence of stimulation by specific molecules includingtumor necrosis factor-α (TNF), interleukin-1 (IL-1) and interferon-γ(IFN-γ) (Kume et al., 1992; Springer, 1994). However, the mecha-nisms responsible for the effects of PM2.5-induced oxidative stresson ICAM-1 and VCAM-1 expressions at the surface of endothelialcells have not been fully elucidated.

PM2.5-induced oxidative stress has been considered as a keymolecular mechanism of PM2.5-mediated toxicity (Deng et al.,2013). Oxidative stress is caused by an imbalance betweenproduction of reactive oxygen species (ROS) and activity of anti-oxidant mechanisms (Limon-Pacheco and Gonsebatt, 2009). ROSare comprised of oxygen radicals such as the superoxide anion(O2

·-), alkoxyradicals (RO·), peroxy radicals (ROO·) and hydroxyl radical(·OH) as well as non-radical forms like hydrogen peroxide (H2O2) orother hydroperoxides (ROOH). A study by Kunsch and Medford(1999) has implicated ROS as signal transduction agents in strainedendothelium.

Exposure of cells to ROS or ROS generation systems has longbeen known to be capable of stimulating various cellular signaling

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pathway (Eckers and Klotz, 2009). ROS appear to be involved in theactivation of redox-sensitive signal transduction such as themitogen-activated protein kinases (MAPKs) and PI3K/AKT path-ways (Harris and Shi, 2003; Huang et al., 2011; Naik and Dixit,2011). MAPK has been implicated in cardiovascular disease (Wadaand Penninger, 2004; Panka et al., 2006). MAPK is a group of pro-tein Serine/threonine kinases that are activated in response to avariety of extracellular stimuli and mediate signal transductionfrom the cell surface to the nucleus, including c-Jun NH2-terminalkinases ( JNK), extracellular signal-regulated kinases (ERK) and p38MAPK (Davis, 2000). ERKs, AKT kinases, stress-activated proteinkinases and nuclear factor-kappa B (NF-κB) are the potentialtargets of ROS in endothelial cells (Singh et al., 2002). Among them,ERKs are important mediators of cell proliferation. JNK and p38MAPK as stress-activated protein kinases are involved in apoptosis.p38 MAPK has been implicated in the upregulation of ICAM-1resulting in endothelial dysfunction. NF-κB are involved in endothe-lial dysfunction and vascular inflammation (Gilmore, 2006), whereasAKT kinases has been associated with antiapoptotic signaling andare regulated by ROS in the angiotensin II-stimulated smoothmusclecells (Irani, 2000). Therefore, ROS serve as important cellular signalingmechanisms that may be responsible for the induction of vascularlesions and subsequent atherosclerosis (Singh et al., 2002).

Using 99mtechnetium-labeled carbon particles, which are verysimilar to the ultrafine fraction of actual pollutant particles,Nemmar et al. (2002) demonstrated that inhaled particles pass rap-idly into the systemic circulation in healthy volunteers, suggestingdirect impacts on the heart and vessels of particulate air pollution.In the present study, using a human endothelial cell line, EA.hy926,as an in vitromodel, we addressed the question of whether PM2.5-induced ROS generation affects ICAM-1 and VCAM-1 expressionsand the adhesion of human monocytic THP-1 cells to the EA.hy926 monolayer. It is known that EA.hy926 cells normally expressICAM-1 and VCAM-1, so this cell line may be useful model to studythe interactions of cardiovascular endothelial cells with stressfulstimuli (Yu et al., 2010; Wei et al., 2011; Yi et al., 2012; Napierskaet al., 2013). The involvement of ROS-dependent JNK, ERK1/2,p38 MAPK, AKT phosphorylation and NF-κB activation were alsoevaluated using selective kinase inhibitors.

Materials and Methods

Materials

A human umbilical vein cell line (EA.hy926) cells were obtainedfrom the China Center for Type Culture Collection (Shanghai,China). Human monocytic leukaemia cell line THP-1 cells wereobtained from the Cell Bank of Peking Union Medical College(Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM),Roswell Park Memorial Institute (RPMI) 1640 medium andpenicillin/streptomycin were purchased from Gibco (Grand island, NY,USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), dimethylsulfoxide (DMSO), 2’,7’-dichlorodihydrofluoresceindiacetate (DCFH-DA) were purchased from Sigma (St Louis, MO,USA). SP600125, SB203580, BAY11-7082 and LY294002 wereobtained from Beyotime (Shanghai, China). U0126 was obtainedfrom Promega (Madison, WI, USA). Fetal bovine serum was pur-chased from PAA (Linz, AUS). Twenty-four well, 6-wells, 96-wellsplates and cell culture dishes were obtained from CostarCambridge (Tewksbury, MA, USA). FITC-anti-ICAM-1 and PE-anti-VCAM-1 antibody were purchased from Biolegend (San Diego,CA, USA). JNK, ERK1/2, p38 MAPK, AKT, p65, phospho-JNK,

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phospho-ERK1/2, phospho-p38, phospho-AKT antibodies werepurchased from Bioworld (Louis Park, MN, USA).

Particle Collection and Preparation

PM2.5 samples were collected continuously at Yuquan Road,Beijing, China in September to October 2012. The sampling sitewas located in University Campus, about 20m from a streetcharacterized by moderate traffic and commercial activities. Theresidential area is characterized by low traffic density. In thesurrounding areas, large industrial and thermoelectric plants wereabsent. Sampling inlets were installed at a height of 10m abovethe ground. PM2.5 samples were collected on Teflon filters(diameter=47mm; Whatman, Piscataway, NJ, USA) for biologicalassay using a low volume samplers (42 lmin–1, URG, Chapel Hill,NC, USA) for 12h (08:00 to 20:00 hours). Before or after sampleswere collected, the Teflon filters were equilibrated in a conditionof 30% relative humidity and 25 °C room temperature for over48h and then weighted on a high-precision microbalance(AG258; Mettler Toledo, Columbus, OH, USA) to measure the massof collected PM2.5. All sampled filters were stored in the darkness at–20 °C before further chemical and physical characterization.Blanks (unexposed filters) were prepared using the same methodas for the samples except for sampling, which were used as acontrol in all experiments.PM2.5 samples on Teflon filters were extracted according to the

method of Imrich et al. (2000). Briefly, Teflon filters were put into a50-ml centrifuge tube, probe-sonicated for 1min in ultra-pure(18.2 MΩ/cm) water, the were then dried filters by drying oven,equilibrated for 48 h and weighed on a microbalance. Accordingthe weight decrease of filters, we qualified the mass of particleswhich suspend in water. The extract particles were put together,andmade the concentration at 5mgml–1, then stored at –80 °C untilphysical and chemical characterization analysis or cell exposure.

PM2.5 Physical and Chemical Characterization

The size distribution of PM2.5 was performed by scanning electronmicroscopy (SEM, JSM-6700F, JEOL, JAPAN) at a magnification of30 000× and accelerating voltage of 5 kV. After being dried over-night, the samples were fixed onto stubs by double-sided tapeand sputter coated with gold for scanning electron microscope(SEM) observation. The size distribution of PM2.5 in suspensionwas analyzed using a Nano-Zetasizer (1000 HS; Malvern Instru-ment Ltd., Worcestershire, UK) based on a dynamic light scatteringmeasurement technique. Before measurement, PM2.5 was firstlysuspended in serum-free culture medium, and sonicated for 30 sat 40W in a bath to disperse the PM2.5 using an ultrasonic processor(VCX130, Sonics, Newtown, CT, USA). The particle Z-Average wasreported.Inorganic elements, organic and elemental carbon (OC and EC),

water soluble inorganic ions and polycyclic aromatic hydrocarbons(PAHs) were detected in our extract PM2.5 samples. 200μl (1mg)PM2.5 samples were used for inorganic elements detection by aciddigestion (HNO3:HF=7:3), followed by measurement using induc-tively coupledplasma-mass spectrometry (ICP-MS, Thermo, ElementalX7, Waltham, MA, USA) for concentrations of Ti, V, Cr, Mn, Co, Ni, Cu,As, Sr, Mo, Cd, Cs, Ba, Pb, Zn, Fe, Ca, K, Mg and Na. The water solubleinorganic ion composition (SO4

2-, NO3- , NH4

+ and Cl-) was determinedwith ion chromatography (Dionex-600, Sunnyvale, CA, USA).PM2.5 samples were frozen drying to powder to detect OC/EC

and PAHs. PM2.5 powder were put on the quartz filters which

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preheated at 450 °C for 4 h, then EC and OC in PM2.5 weremeasured on-filter using a thermal-optical analyzer (SunsetLaboratories, Hillsborough, NC, USA) according to the ACE-Asiabase case protocol as described in the methods of Zhanget al. (2008). PAHs was measured by a thermal desorption at300 °C coupled with cold trapping, and followed by gaschromatography-mass spectrometry (GC/MS) analyzes (Model6890N; Aglient, Santa Clara, CA, USA) equipped with a 60-mHP-5MS column as described by Zhang et al. (2009). FifteenPAHs were analyzed in the PM2.5 samples including acenaphthylene,acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene,pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[123-cd]pyrene, dibenz[a,h]anthracene and coronene.

Cell Culture and PM2.5 Treatment

Human umbilical vein EA.hy926 cells were cultured in high glucoseDMEM supplemented with 10% (v/v) FBS, 100 IUml–1 penicillinand 100μgml–1 streptomycin at 37 °C in a 5% CO2 humidifiedatmosphere. All cell exposure experiments were performed at80–90% of cell confluence with viability≥ 90% determined bythe trypan blue staining. The cells were harvested using 0.25%trypsin and sub-cultured into 6-well plates, 24-well plates, or96-well plates according to the selection of experiments. Cellswere allowed to attach the surface in DMEM supplemented 10%FBS for 24 h prior to treatment. Then, the culture medium wasreplaced with serum-free medium and the cells were incubatedwith freshly dispersed PM2.5 preparations at a final concentrationof 25, 50, 100 or 200μgml–1 for 1, 3, 6, 12 or 24h at 37 °C with orwithout NAC (5mM) pretreatment, respectively. For the inhibitoryeffect experiments, the cells were pretreated with or without inhi-bitors of JNK (SP600125, 25μm), ERK (U0126, 10μm), p38 MAPK(SB203580, 25μm) and PI3K/AKT (LY294002, 25μm) or NF-κB(BAY11-7082, 5μm) and then treated with PM2.5 for the indicatedconcentration and duration, respectively.

Human monocytic leukaemia cell line THP-1 cells were culti-vated in suspension in RPMI-1640 medium containing 10% FBS,100 IUml–1 penicillin and 100μgml–1 streptomycin at 37 °C in 5%CO2 and 95% air humidified atmosphere.

MTT Assay

Cell viability was assessed using the MTT assay (Wei et al., 2011).The EA.hy926 cells were plated into 96-well plates at a density of1.0 × 104 cells per well in 100μl of medium and cultured for 24 h.After incubation, EA.hy926 cells were treated with 0, 25, 50, 100and 200μgml–1 PM2.5 for 1, 3, 6, 12 and 24h, respectively. Afterthe exposure, 20μl of MTT (0.5mgml–1 in PBS) was added to eachwell and incubated for 1 h at 37 °C. Cells were then treated with100μl of dimethyl sulfoxide (DMSO). The absorbance was quanti-fied at 492nm by a microplate spectrophotometer (Thermo MK3,Waltham, MA, USA). The viability of the treated cells was expressedas a percentage of untreated cells, whichwas assumed to be 100%.

Reactive Oxygen Species Assay

The level of intracellular ROS generation was determined bymeasuring the oxidative conversion of DCFH-DA to fluorescentcompound dichlorofluorescin (DCF). Thus, the intracellular ROSgeneration of cells can be investigated using the DCFH-DA as anindicator to detect and quantify intracellular produced reactive

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oxygen species (Wan et al., 1993; Deng et al., 2013). Briefly, EA.hy926 cells (2× 105 cells) were treated with 25, 50, 100 and200μgml–1 PM2.5 for 1, 3, 6, 12 and 24h, respectively. The cellswere lysed with 400mM of NaOH. The fluorescence intensity wasdetected in a fluorescencemulti-well plate reader (Berthold, TriStarLB 941, Baden-Württemberg, Germany) with excitation andemission wavelengths of 488 nm and 530nm. Results weremeasured as the fluorescence intensity change of untreated cells.

Western blotting analisis

After exposure to PM2.5, EA.hy926 cells were separated from theculture medium and lysed in RIPA buffer (50mM Tris-HCl, pH 8.0,150mMNaCl, 1% Triton X-100, 0.5% sodium deoxycholate supple-mented with protease inhibitor cocktail) and phosphotase inhi-bitor cocktail at 4 °C for 30min. Whole cell lysates were collectedusing centrifugation at 12 000 g for 5min at 4 °C. Nuclear proteinswere purified using a nuclear and cytoplasmic protein extractionkit (Beyotime, Beijing, China) according to the manufacturer’sinstruction. Then proteins were subjected to 10% sodium dodecylsulfate-polyacrylaminde gel electrophoresis (SDS-PAGE). The gelswere transferred to a PVDF membrane by semidry electrophoretictransfer at 1mA/cm2 for 60min. The PVDF membranes were thenblocked in 5% non-fat milk at room temperature for 1 h and thenincubated with the primary antibodies diluted (1:1000) inTris/buffered saline/Tween-20 (TBST) containing 5% bovine serumalbumin for overnight in 4 °C, and then incubated with the secon-dary antibody diluted (1:1000) at room temperature for 1 h. Themembranes were washed with TBST four times for 5min each.Immunoreactive bands were detected with ECL reagents(Millipore, Billerica, MA, USA) according to the manufacturer’sinstructions. β-Actinwas used as loading controls for the total proteincontent and showed no differences between groups.

Analysis for Adhesion Molecule Expression

The levels of ICAM-1 and VCAM-1 expression in EA.hy926 cellswere detected using flow cytometry analysis as previouslydescribed (Ali et al., 2004; Huang et al., 2014). After EA.hy926cells were exposed to 0, 25, 50 or 100μgml–1 PM2.5 for 24h or100μgml–1 PM2.5 for 1, 3, 6, 12 and 24h, respectively. Cellswere harvested from plates with trypsin and centrifuged at 800 gfor 5min. The pellet was labeled with anti-ICAM-1 antibody (1:50dilution) or anti-VCAM-1 antibody (1:50 dilution) in PBS supple-mented with 0.3% of bovine serum albumin (PBSA) at 4 °C for30min in the dark. Subsequently, the labeled cells were rinsedtwice with cold phosphate-buffered saline (PBS) in microfugetubes and centrifuged at 800 g for 5min to remove anyunattached antibody. ICAM-1 and VCAM-1 expression weremeasured by a flow cytometer (Beckman FC500, Brea, CA, USA)and expressed as the mean fluorescence intensity.

Preparation of Fluorescent THP-1 Cells

To visualize the THP-1 cells adherent to the EA.hy926 cells, fluores-cent THP-1 cells were prepared according to the method of Kinardet al. (2001). In brief, the THP-1 cells were labeledwith a fluorescentdye, calcein-AM, by incubating 1.0× 107 cells in 4ml of RPMI-1640containing 10% FBS and 40μl of calcein-AM to make a finalcalcein-AM concentration of 10μM at 37 °C for 1 h. Dye loadingwas stopped by adding 4ml of cold RPMI-1640 containing 10%FBS to the suspension and then centrifuging. Fluorescence-labeled

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PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB pathway

cells were resuspended in 4ml of RPMI-1640 containing 10% FBSand prepared for use in adhesion experiments.

Adhesion Experiment of THP-1 Cells

To investigate the effect of the concentration of PM2.5 added to acell culture medium on the adhesion of THP-1 cells to EA.hy926cells, cells were exposed to 25, 50 or 100μgml–1 PM2.5 for 24h or100μgml–1 PM2.5 for 1, 3, 6, 12 and 24h, respectively. For theinhibitory effect experiments, the cells were pretreated with orwithout inhibitors of JNK (SP600125, 25μm), ERK (U0126, 10μm),p38 MAPK (SB203580, 25μm) and PI3K/AKT (LY294002, 25μm) orNF-κB (BAY11-7082, 5μm) and then treated with 100μgml–1 PM2.5for 24 h, respectively. The cells in the EA.hy926 culture wererinsed three times with 1ml of PBS (pH 7.3) to wash out thePM2.5. Then 500μl of new culture medium containing antibioticswas put in each well. Then 20-30μl of suspension of calcein-AM-labeled THP-1 cells (5.0 × 105 cells) was added to each culturewell. They were incubated for 1 h at 37 °C in 5% CO2 and 95%air humidified atmosphere. After that, the suspension of THP-1 cellsin each well was discarded, and the EA.hy926 culture was gentlywashed three times with 1ml of PBS (pH7.3) in order to removenon-adherent THP-1 cells. Adherent fluorescence-labeled THP-1 cellswere observed under a fluorescence microscope (Nikon, Eclipse Ti,Japan) (Kim et al., 2007). The fluorescence intensity of the adherentTHP-1 cells was determined using a Multi-well Plate Reader withexcitation and emission wavelengths of 488 and 530nm.

Figure 1. PM2.5 size distribution. (A) Scanning electron microscope (SEM)images of PM2.5 (Bar, 0.1 μm;magnification 30 000×). (B) PM2.5 size distribu-tion in the Dulbecco’s Modified Eagle’s Medium (DMEM) culture mediumwas detected by dynamic light scattering.

Table 1. Metals detected in PM2.5

Metals Concentration (ngm–3)

Ti 56.58± 2.04V 2.66± 0.05Cr 8.61± 0.18Mn 51.71± 1.02Co 0.55± 0.01Ni 3.93± 0.06Cu 26.62± 0.51As 14.31± 0.38Sr 11.10± 0.16Mo 1.39±0.03Cd 2.87± 0.05Cs 0.86± 0.01

Statistical Analysis

Data are expressed as the means ± SD. The differences betweenthe mean values of two groups were determined by Student’st-test. Associations between the different variables were exam-ined by one-way ANOVA, followed by post hoc comparisonsusing the Tukey’s multiple paired comparison test. Statisticalsignificance was set at P< 0.05.

Results

Physichemical Characteristics of PM2.5

The morphology of PM2.5 by SEM was shown in Fig. 1A. Dynamiclight scattering measurement of PM2.5 showed a size range of0.04–5μm with a mean of 0.28μm (Fig. 1B).

Among the total of 20 metal elements measured, Fe, Pb, Zn, Tiand Mn were the most abundant elements in the PM2.5 samples(Table 1). The analytical results of water soluble inorganic ions inPM2.5 showed SO4

2- (12.49± 0.97μgm–3), NO3- (7.18± 0.69μgm–3),

NH4+ (4.78± 0.29μgm–3) and Cl- (4.09 ± 0.35μgm–3), respectively

(Table 2). The average concentrations of OC and EC in thePM2.5 were 18.51± 3.25μgm

–3 and 2.90± 0.07μgm–3, and theOC/EC ratio was 6.39 (Table 3). Acenaphthylene, acenaphthene,benzo[b]fluoranthene, benzo[a]pyrene and chrysene were thedominant PAHs in PM2.5 (Table 4).

Ba 31.09± 0.39

Pb 146±3Zn 253±3Fe 1071±7Ca 1676±16K 1088±6Mg 396±2Na 803±13 51

Effect of PM2.5 on Cell Viability in EA.hy926 Cells

EA.hy926 cells were exposed 0, 25, 50, 100 or 200μgml–1 PM2.5 for1, 3, 6, 12, or 24 h, respectively (Fig. 2). After 1-, 3- and 6-h exposureof cells to 25, 50, 100 and 200μgml–1 PM2.5, no statistically signifi-cant impacts of PM2.5 were detected compared with the unex-posed control cells. However, a dose-dependent decrease of cell

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Table 2. Water soluble inorganic ions detected inPM2.5

Water soluble ions Concentration (μgm–3)

SO42- 12.49± 0.97

NO3- 7.18± 0.69

NH4+ 4.78± 0.29

Cl- 4.09 ± 0.35

Table 3. Carbonaceous fractions detected in PM2.5

OC/EC Concentration (μgm–3)

OC 18.51± 3.25EC 2.90± 0.07OC/EC 6.39± 1.27

Table 4. Organic compositions detected in PM2.5

PAHs Concentration (ngm–3)

Acenaphthene 20.64± 1.12Acenaphthylene 20.40± 0.71Anthracene 3.33± 0.15Benz[a]anthracene 5.23± 0.34Benzo[a]pyrene 8.14± 0.53Benzo[b]fluoranthene 9.33± 0.65Benzo[k]fluoranthene 4.89± 0.15Chrysene 6.78± 0.18Coronene 0.55± 0.02Dibenz[a,h]anthracene 0.61± 0.02Fluoranthene 3.29± 0.27Fluorene 0.73± 0.06Indeno[123-cd]pyrene 5.56± 0.24Phenanthrene 0.07± 0.01Pyrene 3.54± 0.18

Figure 2. Effect of PM2.5 exposure on cell viability in EA.hy926 cells. EA.hy926 cells were treated with 0, 25, 50, 100 or 200 μgml–1 PM2.5 for 1, 3,6, 12 or 24 h, respectively. The cell viability was assayed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Valuesare the mean± SD of three independent experiments. *P< 0.05,**P< 0.01 vs. the untreated control cells.

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viability was observed after 12- and 24-h exposure to 50, 100 and200μgml–1 PM2.5.

Figure 3. Effect of PM2.5 on the reactive oxygen species (ROS) generationin EA.hy926 cells. EA.hy926 cells were exposed to 0, 25, 50, 100 or200 μgml–1 PM2.5 for 1, 3, 6, 12 or 24 h, respectively. The ROS levels weredetermined by measuring the oxidative conversion of DCFH-DA to DCF.To investigate if the PM2.5-induced changes were related to the decreasein the antioxidant defense, EA.hy926 cells were pretreated with5 mM N-acetylcysteine (NAC) for 1 h and then treated with 100 μgml–1 PM2.5 forthe indicated time. Results were measured as mean fluorescence (arbitraryunits, AU). Values are the mean± SD of three independent experiments.*P< 0.05, **P< 0.01, ***P< 0.001 vs. the untreated control cells.#P< 0.05, ##P< 0.01 vs. 100μgml–1 PM2.5.

Effect of PM2.5 on Intracellular ROS Generation in EA.hy926Cells

The potential of PM2.5 to induce intracellular ROS generation in EA.hy926 cells was evaluated by DCFH-DA intensity. The cells weretreated with 0, 25, 50, 100 or 200μgml–1 PM2.5 for 1, 3, 6, 12 or24h, respectively. After 1 h of exposure to the indicated concentra-tion of PM2.5, ROS generation was evident in relation to the variousconcentrations used (Fig. 3). In addition, Fig. 3 shows that the highestROS generation was observed after 12h with a decline thereafter,indicating that ROS generation is dose dependent. However,increased ROS induced by PM2.5 exposure (100μgml

–1) was signifi-cantly attenuated by pretreatment with NAC.

Effect of PM2.5 on ICAM-1 and VCAM-1 Expressions on EA.hy926 Cells

In order to establish the role of PM2.5-induced oxidative stress, wemeasured ICAM-1 and VCAM-1 cell surface expressions in responseto PM2.5. As shown in Fig. 4, both ICAM-1 and VCAM-1 cell surfaceexpressions in EA.hy926 cells were significantly increased in a

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dose- and time-dependent manner (Fig. 4A and B). However, theincreases in ICAM-1 and VCAM-1 cell surface expressions inresponse to PM2.5 were attenuated by NAC (Fig. 4A). These resultsindicated that PM2.5-induced ROS are capable of stimulating CAMexpression at the cell surface in EA.hy926.

Effect of PM2.5 on THP-1 Cell Adhesion to EA.hy926 Cells

To investigate the effect of PM2.5-induced oxidative stress on theadhesion of THP-1 cells to the surface of EA.hy926 cells, we

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Figure 4. Effect of PM2.5 on intercellular adhesion molecule-1 (ICAM-1)and vascular adhesion molecule-1 (VCAM-1) expressions in EA.hy926 cells.EA.hy926 cells were exposed to 0, 25, 50 or 100 μgml–1 PM2.5 for 24 h (A),or 100 μgml–1 PM2.5 for 1, 3, 6, 12 or 24 h (B). The ICAM-1 and VCAM-1 cellsurface expressions were detected by flow cytometer as described in theMethods section. To investigate if the PM2.5-induced reactive oxygen spe-cies (ROS) generation was related to the increase expression of cell adhe-sion molecule (ICAM-1 and VCAM-1) in the cell surface, EA.hy926 cellswere pretreated with 5mMN-acetylcysteine (NAC) for 1 h and then treatedwith 100μgml–1 PM2.5 for the indicated time. Values are the mean ± SD ofthree independent experiments. *P< 0.05, **P< 0.01 vs. the untreatedcontrol cells. ##P< 0.01 vs. 100 μgml–1 PM2.5.

PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB pathway

measured the fluorescence intensity of adherent THP-1 cells in theEA.hy926 culture after incubation with THP-1 cells in a culture me-dium containing PM2.5 at 0, 25, 50 or 100μgml

–1 PM2.5 for 24 h or100μgml–1 PM2.5 for 1, 3, 6, 12 and 24h. Figure 5 summarizes theresults. As evident from the figure, the adhesion of THP-1 cells wasgreatly enhanced by the presence of PM2.5 under a monolayer ofEA.hy926 cells. At any PM2.5 concentration and any time, thenumber of adherent cells in the EA.hy926 culture was significantlyincreased. However, PM2.5-induced increases in the number ofadherent cells were significantly attenuated when EA.hy926 cellswere pretreated with NAC (Fig. 5A). These results suggested thatPM2.5-induced ROS was involved in promoting the adhesion ofmonocytes to the endothelial cells.

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Effect of PM2.5 on ROS-Dependent Signaling Pathways in EA.hy926 Cells

To assess whether JNK, ERK1/2, p38 MAPK, AKT and NF-κB signal-ing pathways were involved in PM2.5-induced ROS generation, wefirstly detected the phosphorylation of JNK, ERK1/2, p38 MAPK and

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AKT as well as the activation of NF-κB after exposure of EA.hy926cells to 0, 25, 50 or 100μgml–1 PM2.5 for 24h. As shown in Fig. 6A,PM2.5 markedly triggered an increase in ERK1/2 and p38 MAPKphosphorylation from 25 to 100μgml–1 PM2.5 after exposure.Phosphorylations of AKTwere significantly increased after exposureto 100μgml–1 PM2.5. Moreover, there was a significant increase inNF-κB activation in a concentration-dependentmanner. These datasuggested that PM2.5 exposure induces phosphorylation of JNK,ERK1/2, p38 MAPK, AKT phosphorylation and activation of NF-κB.To confirm whether PM2.5-induced ROS generation was

involved in the JNK, ERK1/2, p38 MAPK, AKT and NF-κB signalingpathways in EA.hy926 cells, we further detected the phosphoryla-tion of JNK, ERK1/2, p38 MAPK, AKT and activation of NF-κB afterexposure of cells to 100μgml–1 PM2.5 in the presence of NAC(5mM) for the indicated time. As shown in Fig. 6A, phosphoryla-tion of JNK, ERK1/2, p38 MAPK, AKT and activation of NF-κB weresignificantly attenuated after pretreatment with NAC as comparedwith themarked increase in AKT phosphorylation in the absence ofNAC (Fig. 6A). These findings indicated that JNK, ERK1/2, p38MAPK, AKT and NF-κB pathways are involved in ROS generationin response to PM2.5. Moreover, we also found that treatment withPM2.5 increased JNK, ERK1/2, p38 MAPK, AKT phosphorylation andNF-κB activation in a time-dependent manner (Fig. 6B).

Effect of ERK1/2, JNK, p38 MAPK, AKT and NF-κB Inhibitors onPM2.5-Induced ICAM-1 and VCAM-1 Expressions on EA.hy926Cells

To explore the underlying mechanism of JNK, ERK1/2, p38 MAPK,AKT and NF-κB signaling pathways involved in PM2.5-inducedCAM cell surface expression and adhesion of monocytes to the en-dothelium, EA.hy926 cells were next utilized JNK, ERK1/2, p38MAPK, AKT and NF-κB inhibitors after exposure of EA.hy926 cellsto 100μgml–1 PM2.5 for 1 h. Phosphorylations of JNK, ERK1/2, p38MAPK, AKT and N-p65 were significantly inhibited by the indicatedinhibitors, respectively (data not shown). As shown in Fig. 7, pre-treatment with ERK1/2 inhibitor (U0126), AKT inhibitor(LY294002) and NF-κB inhibitor (BAY11-7082) inhibited ICAM-1and VCAM-1 cell surface expressions after exposure to PM2.5(P< 0.001). However, JNK inhibitor (SP600125) and p38 MAPKinhibitor (SB203580) did not affect on ICAM-1 and VCAM-1 cell sur-face expressions. These data suggested that ERK/AKT and NF-κBpathways mediate PM2.5-induced ICAM-1 and VCAM-1 cell surfaceexpressions in EA.hy926 cells.

Effect of ERK1/2, JNK, p38 MAPK, AKT and NF-κB Inhibitors onPM2.5-Induced THP-1 Cell Adhesion to EA.hy926 Cells

ICAM-1 and VCAM-1 are involved in the adhesion mechanismsthat underlie interactions between endothelial cells with other celltypes. We previously showed that PM2.5 increases the expressionof these adhesion molecules on EA.hy926 cells, and thus to evalu-ate the functional significance of the observation, we assessed theadhesion of THP-1 cells to EA.hy926 cells after pretreatment withJNK inhibitor (SP600125), ERK1/2 inhibitor (U0126), p38 MAPKinhibitor (SB203580), AKT inhibitor (LY294002) and NF-κB inhibitor(BAY11-7082). As shown in Fig. 8, the adhesion of THP-1 cells wasnot affected by pretreatment with JNK inhibitor (SP600125) andp38 MAPK inhibitor (SB203580) in terms of fluorescence intensityas compared to PM2.5-treated cells. However, the fluorescenceintensity markedly appeared to decrease after pretreatment withERK1/2 inhibitor (U0126), AKT inhibitor (LY294002) and NF-κB

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Figure 5. Effect of PM2.5 on THP-1 cell adhesion to EA.hy926 cells. (A and B) Representative fluorescence microscopic photographs of Ca-AM-labeled THP-1cell adherent to EA.hy926 cells. (C andD) Quantitative data from the three independent experiments are shown in A and B. EA.hy926 cells were exposed to 0,25, 50 or 100 μgml–1 PM2.5 for 24 h (A), or 100μgml

–1 PM2.5 for 1, 3, 6, 12 or 24 h (B). EA.hy926 cells were incubated with Ca-AM-labeled THP-1 cells for 1 h,and then washed three times with cold PBS to remove the non-adherent THP-1 cells. The intensity of adherent fluorescence-labeled THP-1 cells was ob-served using a fluorescence microscope as described in the Methods section. The fluorescence intensity was detected using a multi-well plate reader withexcitation and emission wavelengths of 488 nm and 530 nm. To investigate if the PM2.5-induced reactive oxygen species (ROS) generation were related tothe increase THP-1 cells adhesion to EA.hy926 cells, EA.hy926 cells were pretreated with 5mM of N-acetylcysteine (NAC) for 1 h and then treated withμg ml–1 PM2.5 for the indicated time. Values are the mean ± SD of three independent experiments. *P< 0.05, **P< 0.01 vs. the untreated control cells.##P< 0.01 vs. 100 μgml–1 PM2.5.

W. Rui et al.

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Figure 6. Effect of PM2.5 on reactive oxygen species (ROS)-dependent ERK1/2, JNK, p38 MAPK, AKT phosphorylation and NF-κB activation in EA.hy926 cells.EA.hy926 cells were exposed to 0, 25, 50 or 100μgml–1 PM2.5 for 24 h (A), or 100μgml

–1 PM2.5 for 1, 3, 6, 12 or 24 h (B). Cell lysates were blotted for phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, phospho-p38 MAPK, p38 MAPK, phospho-AKT, AKT, and nuclear and cytoplasmic protein extraction of N-p65 and C-p65were blotted, respectively. Density of phosphorylated-JNK/ERK1/2/P38 MAPK/AKT and N-p65 was relative to the level of JNK/ERK1/2/P38 MAPK/AKTand C-p65, respectively. To investigate if the PM2.5-induced ROS generation were related to JNK, ERK1/2, p38 MAPK, AKT and NF-κB signaling pathways,EA.hy926 cells were pretreated with5 mM N-acetylcysteine (NAC) for 1 h and then treated with 100 μgml–1 PM2.5 for the indicated time. Values are themean ± SD of three independent experiments. *P< 0.05, **P< 0.01 vs. the untreated control cells. #P< 0.01 vs. 100 μgml–1.

PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB pathway

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inhibitor (BAY11-7082). These findings suggested that ERK/AKTand NF-κB pathways are involved in the adhesion of THP-1 cellsto EA.hy926 cells in response to PM2.5.

DiscussionThe present study demonstrates that PM2.5 induced an increase inintracellular ROS generation, followed by upregulation JNK,ERK1/2, p38 MAPK and AKT phosphorylation, and nuclear translo-cation of NF-κB in human EA.hy926 cells in a dose- and time-dependent manner. The cell surface expressions of adhesionmolecules, ICAM-1 and VCAM-1, and the adhesion of THP-1 cellto EA.hy926 cells were also significantly increased after the PM2.5exposure. In contrast, scavenging ROS with NAC significantlyattenuated the cell surface expression of adhesion molecules,decreased cell adhesion and markedly reduced ROS-dependentkinases phosphorylation. Moreover, pretreatment with ERK1/2 in-hibitor (U0126), AKT inhibitor (LY294002) and NF-κB inhibitor

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(BAY11-7082), but not a JNK inhibitor (SP600125) or a p38 MAPKinhibitor (SB203580), significantly inhibited PM2.5-induced ICAM-1and VCAM-1 cell surface expressions on EA.hy926 cells and adhe-sion of THP-1 cells to EA.hy926 cells. Taken together, these resultssuggest that PM2.5-induced ROS may contribute to ICAM-1 andVCAM-1 expressions by activating the ERK/AKT/NF-κB signalingpathway in the endothelial cells.Many previous studies have demonstrated that chemical com-

position of PM2.5 is involved in toxicological effects, including bothorganic and inorganic fractions (Araujo et al., 2008; Wei et al., 2011;Deng et al., 2013) . Global population-weighted PM2.5 compositioncontains organic mass (32%), mineral mass (30%), sulfate (17%),black carbon (7%), ammonium (7%), nitrate (6%) and sea salt(1%) (Philip et al., 2014). Consistent with our previous study (Denget al., 2013), the study demonstrates that the high levels of Fe, Pb,Zn, Ti, Mn, acenaphthylene, acenaphthene, benzo[b]fluoranthene,benzo[a]pyrene and chrysene are major constituents of PM2.5. Inaddition to metals, PAHs, OC and EC, PM2.5 also appeared to have

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Figure 7. Effect of ERK1/2, JNK, p38 MAPK, AKT and NF-κB inhibitors onPM2.5-induced intercellular adhesion molecule-1 (ICAM-1) and vascular ad-hesion molecule-1 (VCAM-1) expressions on EA.hy926 cells. EA.hy926 cellswere pretreated with U0126 (10 μM), SP600125 (25 μM), SB203580(25 μM), LY294002 (25μM) and BAY11-7082 (5μM) for 1 h and then treatedwith 100 μgml–1 PM2.5 for 24 h, respectively. Cells were processed for flowcytometer analysis as described in the Methods section. Values are themean± SD of three independent experiments. *P< 0.05, **P< 0.01 vs.the untreated control cells. #P< 0.01, ##P< 0.001 vs. PM2.5.

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higher constitutes of water soluble inorganic ions such as SO42- and

NO3- . All these results obtained from the chemical characterization

reflect that PM2.5 samples in Beijing are a complex mixture ofchemicals and a significant contribution from anthropogenicsource. These constituents can play an important role in mediatingcardiovascular effects of PM air pollution (Peters et al., 1997; Popeet al., 2004; Laden et al., 2006; Alfaro-Moreno et al., 2007; Riediker,2007; Araujo et al., 2008).

The components of PM2.5, including transitionmetals, water sol-uble inorganic ions, carbonaceous fractions and polycyclic aro-matic hydrocarbons, caused oxidative stress which have beendescribed as a common pathway for PM2.5-indcued oxidativedamage (Gilmore, 2006; Eckers and Klotz, 2009; Ying et al., 2009;Wei et al., 2011; Feng et al., 2013). In the present study, we foundthat different concentrations of PM2.5 significantly caused a de-crease in cell viability (Fig. 2). It has been reported that PM2.5 anddiesel exhaust particles (DEP) induces intracellular ROS generationin cultured human umbilical vein endothelial cells (Wei et al., 2011)and the microvascular endothelium (Frikke-Schmidt et al., 2011;Wauters et al., 2013). Similarly, we also clearly observed thatPM2.5 induced ROS generation in EA.hy926 cells in a time- anddose-dependent manner. Moreover, NAC, a ROS scavenger, signif-icantly attenuated PM2.5-induced ROS generation (Fig. 3), indicat-ing that PM2.5 is capable of inducing the cytotoxic effect ofoxidative stress induced by ROS.

Previous studies have shown that ROS act as chemical messen-gers in the signal transduction process to activate cell adhesionmolecule expression in endothelial cells (Ying et al., 2009; Frikke-Schmidt et al., 2011; Hemmingsen et al., 2011; Wauters et al.,2013; Wei et al., 2011). There is growing evidence that PM increasesthe adhesion factors expression in endothelium (Montiel-Davaloset al., 2007; Yatera et al., 2008; Li et al., 2010; Lee et al., 2012) . In

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the present study, our results showed that PM2.5 significantlyincreased ICAM-1 and VCAM-1 cell surface expressions and theadhesion of THP-1 cells to EA.hy926 cells in a time- andconcentration-dependent manner (Figs. 4 and 5). However, pre-treatment with NAC significantly reduced PM2.5-induced adhesionfactors cell surface expressions and the adhesion of THP-1 cells toEA.hy926 cells, indicating that PM2.5-induced ROS was involved instimulating the expression of adhesionmolecules and recruitmentof inflammatory cells.

MAPK or AKT is an important regulator of cell growth, survival,proliferation, inflammatory and immune reaction in response tooxidative stress. (Cowan and Storey, 2003; Wan et al., 2010). JNK,ERK and p38 MAPK are involved in stimulation of adhesion factorsexpression in different kinds of cells by different stress stimuli(Chen et al., 2011; Feng et al., 2013; Olejarz et al., 2014). In thepresent study, we evaluated if the JNK, ERK, p38 MAPK and AKTpathways involved in regulating the expression of adhesion mole-cules and recruitment of inflammatory cells in response to PM2.5-induce oxidative stress. Here we found that JNK, ERK1/2, p38MAPKand AKT pathways were activated after exposure to PM2.5 for theindicated times. However, treatment with U0126 and LY294002significantly inhibited ICAM-1 and VCAM-1 cell surface expressionsand the adhesion of THP-1 cells to EA.hy926 cells after exposure toPM2.5, whereas SP600125 or SB203580 had no significant effects.Thus, we propose that ERK/AKT pathway is required for the expres-sion of adhesion molecules and recruitment of inflammatory cells.In addition, we also observed that NAC markedly suppressed thephosphorylations of ERK and AKT after PM2.5 exposure, even whenthe oxidative stress is decreased. The complementary evidenceindicated that ROS generation is associated with the activation ofthe ERK/AKT pathway, which contributes to adhesion moleculesexpression after PM2.5 exposure. These observations are consistentwith the previous reports that ERK/AKT signaling pathway isimplicated in ICAM-1 and VCAM-1 expressions in human aorticendothelial cells (HAECs) (Chen et al., 2011; Feng et al., 2013;Olejarz et al., 2014).

The transcription factor NF-κB regulates a wide variety of biolog-ical effects on stress and adaptive responses in various cell typesand organs ( Jones et al., 2005). Multiple signaling pathways includ-ing MAPK, PKC, PKA, and AKT pathways act directly to phosphory-late NF-κB subunits to affect the ability of NF-κB to bind DNA andincrease the transactivation of NF-κB-dependent genes ( Joneset al., 2005). Thus, the activation of NF-κB and the subsequentincrease in cell adhesion molecule expression have been linkedto the intracellular redox state (Khan et al., 1996; Kunsch andMedford, 1999). Here, our data also showed that NF-κB is activatedand p65 translocated into nucleus after exposure to PM2.5.However, activation of NF-κB was significantly attenuated afterpretreatment with NAC. Moreover, BAY11-7082 inhibited ICAM-1and VCAM-1 cell surface expressions and the adhesion of THP-1to EA.hy926 cells after exposure to PM2.5. These observations areconsistent with the previous reports that NF-κB is responsible forthe ICAM-1 and VCAM-1 expression (Verna et al., 2006; Chenet al., 2011; Mehla et al., 2011; Yi et al., 2012; Feng et al., 2013;Olejarz et al., 2014) .

In conclusion, PM2.5-induced oxidative stress induces the cellsurface expression of ICMA-1 and VCAM-1 through activating theERK/AKT/NF-κB signaling pathway, promotingmonocyte adhesionto endothelial cells. Understanding the signaling pathways impli-cated in PM2.5-mediated oxidative stress provides new insightsinto the pathophysiology of cardiovascular diseases associatedwith airborne pollution.

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Figure 8. Effect of ERK1/2, JNK, p38MAPK, AKT and NF-κB inhibitors on PM2.5-induced THP-1 cell adhesion to EA.hy926 cells. EA.hy926 cells were pretreatedwith U0126 (10 μM), SP600125 (25 μM), SB203580 (25μM), LY294002 (25μM) and BAY11-7082 (5μM) for 1 h and then treated with 100 μgml–1 PM2.5 for24 h, respectively. The fluorescence intensity was detected in a fluorescence multi-well plate reader with excitation and emission wavelengths of 488 and530 nm. The intensity of adherent fluorescence-labeled THP-1 cells was observed under a fluorescence microscope. The cells were incubated withCa-AM-labeled THP-1 cells for 1 h, and then washed three times with phosphate-buffered saline (PBS) to remove the unadherent cells. Fluorescing mono-cytes that adhered on the endothelial cell surface were observed under a fluorescencemicroscope as described in theMethods section (A). The fluorescenceintensity was detected using a multi-well plate reader with excitation and emission wavelengths of 488 nm and 530 nm (B). Values are the mean ± SD ofthree independent experiments. *P< 0.05, **P< 0.01 vs. the untreated control cells. ##P< 0.01 vs. PM2.5.

PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB pathway

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Acknowledgments

This work was financially supported by grants from NationalNatural Science Foundation of China (No. 21377127, 11275264,U1432245), and the CAS/SAFEA International Partnership Programfor Creative Research Teams.

Conflict of interestAll authors declare no competing financial interest.

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