Motorcycle exhaust particles up-regulate expression of vascular adhesion molecule-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells

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Highlights

"_{The motorcycle exhaust particle (MEP) induced adhesion between cells of the THP-1 and HUVECs in a time-dependent manner. " MEPs} could induce ICAM-1 and VCAM-1 expression through oxidative stress and NF-jB activation in HUVECs. " Carbon black (CB) nanoparticles with different diameters could induce VCAM-1 and ICAM-1 protein expression. " Polycyclic aromatic hydrocarbons (PAHs) only increased the expression of ICAM-1 but not that of VCAM-1 in HUVECs.

3 February 2012

1

2

Motorcycle

exhaust particles up-regulate expression

of

vascular adhesion

3

molecule-1 and intercellular adhesion molecule-1

in

human umbilical vein

4

endothelial cells

5

Chen-Chen

Lee

a

,

Shih-Hsuan

Huang

b

,

Ya-Ting

Yang

b

,

Yu-Wen

Cheng

c

,

Ching-Hao

Li

b

,

Jaw-Jou

Kang

b,⇑ 6 a

Department of Microbiology and Immunology, School of Medicine, China Medical University, Taichung,Taiwan 7 b

Institute of Toxicology, College of Medicine, National Taiwan University, Taipei,Taiwan 8 c

School of Pharmacy, Taipei Medical University, Taipei,Taiwan 9 10 1 2

a r t i c l e

i n f o

13 Article history: 14 Received 12 October 2010 15 Accepted 23 January 2012 16 Available online xxxx 17 Keywords:

18 Motorcycle exhaust particle 19 Polycyclic aromatic hydrocarbons 20 Cell adhesion molecule 21 Oxidative stress 22 NF-jB 23 2 4

a b s t r a c t

25

Epidemiological studies have shown that there isa strongcorrelation between atherosclerosis and

ambi-26

ent air pollution. In this study, we found that motorcycle exhaustparticles (MEP)induced adhesion

27

between cellsof thehuman monocytic leukemia cellline (THP-1) and humanumbilical vein endothelial

28

cells(HUVECs) ina time-and dose-dependent manner. In addition,MEP treatmentinduced both mRNA

29

and protein expression ofvascular celladhesionmolecule-1 (VCAM-1)and intercellular adhesion

mole-30

cule-1 (ICAM-1)in HUVECs.The IjB degradation andp65 nuclear translocation was found in MEP-treated

31

HUVECs, suggested the involvement of nuclearfactor-jB (NF-jB).MEP-induced VCAM-1 and ICAM-1

32

protein expression was inhibited by NF-jB inhibitor BAY11-7085. Oxidativestress was also involved

33

in thesignaling ofVCAM-1 and ICAM-1 expression. MEP treatment caused hydrogen peroxide and

super-34

oxideformation. Pretreatmentwitha-tocopherol could inhibit MEP-inducedreactive oxygen

intermedi-35

ates generation and suppressed MEP-induced IjB degradation and adhesion molecules expression.

36

Furthermore, the carbon black (CB) nanoparticles with different diameters could induce VCAM-1 and

37

ICAM-1 protein expression; however, polycyclic aromatic hydrocarbons (PAHs) only increased the

38

expression of ICAM-1 but not that of VCAM-1 in HUVECs. In this study, we found that MEPs could induce

39

ICAM-1and VCAM-1expression through oxidative stress and NF-jB activation in HUVECs.

40

Ó 2012 Published by Elsevier Ltd.

41 42

43 1. Introduction

44 Epidemiological studies indicate that peaks of ambient

particu-45 late air pollution are associated with an increase in pulmonary

46 and cardiovascular morbidity and mortality (Peter et al., 2001).

47 The involvement of the cardiovascular system came to berealized 48 when astudy found an association between air concentrations of

49 particulate matter and the number of hospital admissions for

ische-50 mic heart disease and congestive heartfailure (Schwartz and

Mor-51 ris, 1995). Atherosclerosis, a progressive disease characterized by

52 the accumulation of lipids and fibrous elements in the large arteries,

53 is the singlelargest causeof cardiovascular diseases. Activation of

54 endothelial cells by proinflammatory stimuli has been established

55 as an important link between risk factors and the pathologic

mech-56 anisms underlying atherosclerosis (Hansson, 2005). During this

57 process, the expression of cell adhesion molecules (CAMs) is one

58

of the initial responses of activated endothelium during injury or

59

atherosclerotic plaqueformation (Narizhneva et al., 2005). CAMs in-60 duceleukocyte adhesion and transmigration to the subendothelial

61 space (Krieglstein and Granger, 2001; Wood et al., 1993). In larger

62

blood vessels, these processes are critical for foam cell formation

63

and subsequent development of atherosclerotic lesions. They are

64 critical in the microvasculature for leukocyte recruitment and

65

inflammatory response (Gonzalez-Amaro et al., 1998). Both

pro-66

cesses require up-regulation of members of the selectin family, such

67

as P- and E-selectin, and members of the immunoglobulin

super-68

family, most notably, intercellular adhesion molecule-1 (ICAM-1)

69

andvascular celladhesion molecule-1 (VCAM-1). The importance

70

of CAMs in atherosclerosis was investigated by a study on animals

71

with a null mutation. Deficiency of ICAM-1in apolipoprotein E-null 72

mice appears to inhibit the development of atheroscleroticlesions 73 (Collins et al., 2000). In animals lacking domain 4 of VCAM-1, the

74

area of early atherosclerotic lesions is reduced significantly as

com-75

pared with that in controllittermates (Cybulsky et al., 2001).

76

In Taiwan, motorcycles are widely used and are a major source of

77

airborne pollutants. The use of motorcycles, especially those with

78

the 2-stroke engine, was estimated to introduce about 16,000 tons

0887-2333/$ - see front matter Ó 2012 Published by Elsevier Ltd. doi:10.1016/j.tiv.2012.01.021

⇑ Corresponding author. Address: Institute of Toxicology, College of Medicine, National Taiwan University, No.1 Jen-Ai Road, Section 1, Taipei, Taiwan. Tel.: +886 2 23123456x88603; fax: +886 2 23140217.

E-mail address:jjkang@ntu.edu.tw(J.-J. Kang). Q1

Toxicology in Vitro xxx (2012) xxx–xxx

Contents lists available atSciVerse ScienceDirect

Toxicology in Vitro

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t o x i n v i t

79 of total suspendedparticles and15,000 tons of particulate matter

80 (PM) (diameter, <10

l

m; PM10)per year (Environment Protection

81 Agency (EPA), Taiwan, 1994). This concentration of PM10 is consid-82 erably higher(64

lg/m

3_{) than thatin other parts of the world(e.g.,} 83 London,UK, 14

lg/m

3_{; Paris, France,14}

_l

_g/m3_{)(EPA, Taiwan,1996).} 84 Motorcycle exhaustparticles (MEP) contain a carbon black core,

85 absorbing >110different organic compounds, including the C1– 86 C20 chain of hydrocarbon compounds and polycyclic aromatic

87 hydrocarbons(PAHs) (Ueng et al., 2000). Theresults of in vitro

stud-88 ies indicate thatMEPs arecytotoxic (Lee and Kang, 2002),

muta-89 genic (Zhou and Ye, 1997), andgenotoxic (Cheng et al., 2004). It

90 has also been shown thatMEPs impaired thefunction of an isolated

91 rat aorta and induced proinflammatory effects (Lee et al., 2005).

92 However, the environmental and biological impact of MEP remains

93 unclear, especially with regard to the immune and cardiovascular

94 system. Animal studies have shown thata range ofnanoparticulates 95 deliveredby inhalation andinstillation cancross thealveolar–blood 96 barrier (Kreyling et al., 2002; Memmar et al., 2002; Nemmar et al.,

97 2001; Oberdorster et al., 2002). Once circulated, nanoparticles

98 may interact with the vascular endothelium or have a direct effect

99 on atherosclerotic plaques, and cause local oxidative stress and

pro-100 inflammatory effects similar to those that occur in the lung (Brook

101 et al., 2004). MEP contains nanoparticles that may cause

cardiovas-102 cular system damage.

103 Because CAM hasan important role in a variety of pathological

104 conditions, including atherogenesis, and ICAM-1 and VCAM-1

105 expressions can be induced by cytokines or toxic compounds, we

106 investigated theeffects ofMEPs onVCAM-1 and ICAM-1

expres-107 sion in human umbilical vein endothelial cells (HUVECs). In this

108 study, we found that MEP increased the expression of VCAM-1

109 andICAM-1 in HUVECs throughoxidative stress and NF-jB

activa-110 tion and carbonblack alsocontributedto theexpression of these

111 compounds.

112 2. Materials andmethods 113 2.1. Chemicals

114 PAHs, including anthracene, benzo(a)pyrene, chrysene, fluo-115 ranthene, fluorine, naphthalene, phenanthrene, and pyrene, and

116 antioxidants (a-tocopherol, catalase, and ascorbicacid) were

pur-117 chased from Sigma (St. Louis, USA). Fetal bovine serum (FBS),

118 M199 medium, collagenase (type I), were obtained from Gibco

119 BRL (Grand Island, NY,USA). Thecarbon black (CB) particles used

120 in this study had a primary particle size of 15 nm (HIBLACK

121 900 L,surface area,270 m2/g),29 nm(Printex140 V,surface area,

122 90 m2_{/g), 51 nm (Printex G, surface area, 30 m}2_{/g), and 95 nm} 123 (Flammruss 101, surface area,20 m2_{/g) were purchased from} Evo-124 nikDegussa (Germany). Dimethylsulfoxide (DMSO)was used as

125 solvent control.

126 2.2. Motorcycle exhaustparticles (MEP)preparation

127 Motorcycle exhaust particles (MEP) were collected on 0.5-lm

128 quartz fiber filters (Advantec MFS,Inc.,CA)from 50-cm3_Yamaha 129 Cabin engines using 95 octane unleadedgasoline. The sampling

130 apparatus consisted of a 30-cm long by 2.2-cm diameter stainless

131 dilution tube in sequence with a filterholder anda vacuum pump.

132 The engine was run at an idle speed of 150 rpm on an empty load

133 with the vacuum pump set at a flow rate of 20 L/min to collect MEP

134 for 1h twice daily. The temperature of the motorcycle exhaust

135 where it is being collected at the filter was50 °C. Ithas been

deter-136 mined that the original concentrations of motorcycle exhaust

par-137 ticles (mean values) were 118 mg/m3 for PM1, 216 mg/m3 for 138 PM2.5, and 228 mg/m3for PM10(Ueng et al., 2005). The quartz

fil-139

ters with collectedparticles werekept from light and left to dry in

140

a desiccator and repeatedly extractedfour times with methanol

141

under sonication. The methanol was then removed by a vacuum

142

evaporator. The final residues, the MEP, were collected and kept

143

desiccated at20 °C.In total,32.7

l

gMEP was derived from1 L 144 ofYamaha motorcycle exhaust. MEP was dissolved in 100% DMSO.

145

Particle-free MEPs are primarily composed of chemicals that

in-146

clude PAHs by using qualitativeGC–MS analysis (Peng, 1997).

147

2.3. Human umbilical vein endothelialcells (HUVECs) isolation and

148

treatment

149

Human umbilical vein endothelial cells were obtained from the

150 National Taiwan University Hospital and isolated via enzymatic

151

digestion from 20cm-long umbilicalcord vein segments filled with

152

0.1% collagenase (Jaffe et al., 1973).Confluent primarypassages

be-153

tween threeand fivewere used in the experiment. To analyze the

154

effects of antioxidants and inhibitors,2  105_{cells were} pre-incu-155

bated for 30 min with antioxidants or inhibitors

a

-tocopherol 156 (30

l

M);ascorbicacid (100

lM); catalase

(3000 U);Bay11-7085

157 (10

l

M) and then stimulatedwith MEP for different timeperiods.

158 The control cells were treated with 0.1% dimethyl sulfoxide

159

(DMSO).

160

2.4. Reverse transcription polymerasechain reaction(RT-PCR)

161

Total RNA was isolated byacid guanidinium thiocyanate–phe-162 nol–chloroform extraction, resuspended in diethylpyricarbonate

163 (DEPC)-treated water and quantified spectrophotometrically as

164

previous described (Chomczynski and Sacchi, 1987). To analyze cell

165

adhesion molecule mRNA expression,RT-PCR wereperformed by

166

evaluating ICAM-1 and VCAM-1 mRNA content and that of a

167

house-keeping gene, b-actin, as an internal control. In brief, the first

168

strand cDNA was synthesized from 3

lg total RNA at

42 °Cfor

169

60min.Specific ICAM-1 and VCAM-1 cDNAs were amplified as

fol-170

lows: denaturation at95 °Cfor 5min,followed by 35 cycles of

dena-171

turation at95 °Cfor 30s,annealing at60 °Cfor 30s,and extension

172

at72 °Cfor 30s,and a final10 minextension at72 °C.The PCR

prod-173

ucts were separated by 2% agarose gel electrophoresis, with

ethi-174

dium bromidestaining. The primer sets were listed as: ICAM-1

175

(sense,50_{-CCTCACACTTCACTGTCACCT-3}0_{, antisense, 5}0_-CGTGCCGCA 176 CTGAACTGGAC-30_{);VCAM-1(sense, 5}0_{-ATCCCTACCATTGAAGATAC}

177 TGG-30_,antisense,50_{-TGATGACAGTGTCTCCTTCTTTG-3}0_{) (}

Greiffen-178

berg et al., 1997); b-antin (sense, 50_{-TGACCCAGATCATGTTT} GAG-179 30_{, antisense, 5}0_{-TACTGAGGTAGTCAGTCAGG-3}0_{) (Fan et al., 2007).}

180

2.5. Western blotting

181

Total cell lysate, protein concentration determination, SDS– 182 PAGEand immunoblot were performed as described previously

183 (Liao et al., 2009). For immunodetection, the PVDF membrane

184

was blocked in non-fat milk and incubated in TBST with antibodies

185

specific to p65, IkB-a, histone H1, VCAM-1, and ICAM-1 (Santa 186

Cruz, Santa Cruz, CA, USA), and b-actin (Sigma, Saint Louis, MI,

187

USA). For chemiluminescent detection, blots were incubated with

188

HRP-conjugated secondaryantibodies (1:5000in TBST) for 2hat

189

room temperature, followed by ECL detection, according to the

190

manufacturer’s protocol(Perkin–Elmer).

191

2.6. Nuclear extract preparation

192

After incubation with MEP in 100-mm dishes,8  105_{cells were} 193

washed twice with ice-cold PBS. Cells were scraped from the

194 dishes andthe nuclear protein was extracted using a NE-PER

nu-195

clear andcytoplasmic extractionreagents kit according to the

pro-2 C.-C. Lee et al. / Toxicology in Vitro xxx (2012) xxx–xxx

Please cite this article in press as:Lee, C.-C., et al. Motorcycleexhaust particles up-regulate expressionofvascular adhesion molecule-1 and intercellular adhesion molecule-1inhuman umbilical vein endothelial cells. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2012.01.021

196 cedure recommended by the manufacturer (Pierce, Rockford, IL,

197 USA).

198 2.7. Celladhesion assay

199 The assay of cell adhesion wasperformed according aprevious

200 study (Goda et al., 2000). HUVEC (5  103cell/cm2) was cultured in

201 12-well culturedish andtreated with different concentrations of

202

MEP for 24h. Then, THP-1 cells (2  103_cell/cm2_{), the human} 203

monocyticleukemic cellline, were fluorescentlylabeled by incuba-204 tion with calcein-AM and added to each well(2  103_cell/cm2₎

205

withHUVEC for1 h at37 °C. After removalof non-adherent cells

206

by PBS washing, the adhesive THP-1 cell images were captured

207

in 10 random fields by fluorescence microscope (Zeiss) with a

208

monochrome CCD camera using excitation and emission

wave-209

lengths of 488 and 530 nm. Adherence cell numbers were

quanti-Fig. 1. The enhancement of THP-1 cell adhesion to MEP-treated endothelial cell. HUVECs stimulated with 30 or 100lg/ml MEP for 3 or 24 h and were co-incubated with calcein-AM labeled THP-1 monocytic leukemia cell. The 10 ng/ml TNF-awas used as the positive control. The amounts of adherent THP-1 cells were monitored by fluorescent microscopy and quantification by Image J software. Data were expressed as mean ± S.E.M and statistical analysis was performed on the results of at least three independent biological replicates.⁄⁄⁄

p < 0.001, indicate a statistical difference with the control (n P 3). The control cells were treated with 0.1% dimethyl sulfoxide (DMSO).

C.-C. Lee et al. / Toxicology in Vitro xxx (2012) xxx–xxx 3

210 fied by Image Jsoftware. Analyseswere repeated three times over

211 the same region and the results shown are the mean ± S.E.M. of at

212 least three separate experiments.

213 2.8. Reactive oxygen species production measurement

214 The reactive oxygen species (ROS) level was determined using

215 flowcytometry (FACS Calibur,BD Biosciences, San Jose, CA, USA).

216 The 8104 _{cells seeded in 12-well plate were loaded with} 217 10

lM

2,7-dichlorofluorescein (DCFH-DA) or10

lM

dihydroethidi-218 um (DHE)for hydrogen peroxide and superoxide detection,

respec-219 tively. After 10min staining,cells were exposedto vehicleor MEP

220 (or PAHs) for 2hat37 °C incubator.After that, cells were harvested

221 by trypsinization, and the fluorescence was measured by flow

222 cytometry.

223 2.9. Statisticalanalysis

224 All values refer to themean ± S.E.M.of at least three separate

225 experiments. Statistically significant differences among groups

226 were determined usingone-way analysisof variance (Cherepanova

227 et al., 2009) followedbyNewman–Keuls post hoctest. The level of

228 significance was set at p value< 0.05.

229

3. Results

230

3.1. MEP caused adhesion of cells of human monocytic leukemia

231

cell line to HUVECs through increased expression of adhesion

232

molecules

233

First, we detected the effects of MEP on leukocyte adhesion to

234

the endothelium. As shown inFig. 1, after treatment with 30 and

235 100

l

g/ml of MEP, the adhesion of cells of human monocytic

236

leukemia cell line (THP-1) to HUVECs increased in a time and

237

dose-dependent manner. Further, we investigated the expression

238

of adhesionmolecules inHUVECs after treatment with MEP.We 239 isolated RNA and proteins and analyzed VCAM-1 and ICAM-1

240

mRNA and protein expression by reverse

transcription-polymer-241

ase chain reaction (RT-PCR) andWesternblotting. We found that

242 100

l

g/ml of MEP induced ICAM-1 and VCAM-1 mRNA

expres-243

sion in a time-dependent manner; the mRNA expression of

244

VCAM-1 and ICAM-1 was the highest at6and 12 h,respectively 245 (Fig. 2A).

246

The MEP-induced ICAM-1 and VCAM-1 protein expression

247

peaked at 24 h. It was found that 30 and 100

lg/ml of MEP

in-248

duced similar levels of ICAM-1 and VCAM-1 protein expression

249

in HUVEC (Fig. 2B). For 30 and 100

lg/ml of MEP,

the level of

250

VCAM-1 protein expression increased 2.00 ± 0.15-fold and Fig. 2. The effects of MEP on cell adhesion molecules (VCAM-1 and ICAM-1) mRNA and protein induction in HUVECs. (A) MEP (100lg/ml) induced VCAM-1 and ICAM-1 mRNA expression in HUVEC in a time-dependent manner. Histograms represented quantification of RT-PCR normalized with b-actin and compared with the control as the relative induction of VCAM-1 and ICAM-1. (B) MEP (30 and 100lg/ml) treatment induced VCAM-1 and ICAM-1 expression in both dose-and time-dependent manner. TNF-a

(T) 10 ng/ml was used as the positive control. The contents of VCAM-1 and ICAM-1 protein induction were analyzed by densitometry. Data were expressed as mean ± S.E.M. and statistical analysis was performed on the results of at least three independent biological replicates.⁄

p < 0.05,⁄⁄

p < 0.01, and⁄⁄⁄

p < 0.001, indicate a statistical difference with the control (n P 3). The control cells were treated with 0.1% DMSO.

4 C.-C. Lee et al. / Toxicology in Vitro xxx (2012) xxx–xxx

Please cite this article in press as:Lee, C.-C., et al. Motorcycleexhaust particles up-regulate expressionofvascular adhesion molecule-1 and intercellular adhesion molecule-1inhuman umbilical vein endothelial cells. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2012.01.021

251 2.27 ± 0.21-fold, respectively, and the levels of the ICAM-1

252 protein expression increased 2.85 ± 0.23-fold and 2.95 ± 0.24-253 fold,respectively.

254 3.2. MEP-induced VCAM-1 and ICAM-1 expression through NF-jB

255 activation

256 We investigated whether MEP could induce NF-jB activation in

257 HUVECs. Tumor necrosis factor-a (TNF-a), a known activator of

258 NF-jB, induced p65 translocation from thecytoplasm tothe

nu-259 cleus (Fig. 3A) and was used as the positivecontrol. Itwas found

260 that the translocation of p65 into the nucleusoccurred at30 min

261 aftertreatment with30

lg/ml of MEP; this was detected by

immu-262 noblotting of nuclear proteins (Fig. 3A). Moreover,treatment with

263 MEP caused significant increase in IjB-a degradation starting at 264 30 min after treatment with MEP (Fig. 3B). These resultssuggest 265 thatMEP induced NF-jB activation in HUVECs. Pretreatment with

266 10

l

M BAY11-7085 (Yoon et al., 2004), a NF-jB inhibitor,

com-267 pletely inhibited MEP-induced ICAM-1 and VCAM-1 expression

268 for24 h; this wasdetected by immunoblotting of totalproteins 269 (Fig. 3C).

270

3.3. Reactive oxygen intermediates are involved in MEP-induced

271

VCAM-1 and ICAM-1 expression

272

There is increasing evidence showing that oxidative stress may

273

induce VCAM-1 and ICAM-1 expression (Sung et al., 2007). After

274 HUVECs were treated with 30

l

g/ml of MEP for 2 h, the levels

275

of 2,7-dichlorofluorescein diacetate (DCFH-DA) and dihydroethi-276 dium (DHE) expressed in terms of mean fluorescence index

277

(MFI) significantly increased by 40% as compared to the control

278

(Fig. 4A). MEPcaused an increase in the MFI of DCF and DHE,

279

both of which were inhibited bytreatment with 30

l

M

a

-tocoph-280 erol (TOC) but not with 30

l

Mascorbic acid (ASC) and 3000 U

281

catalase (Cat). Next,different antioxidants wereused to

investi-282

gate the possible involvement ofoxidative stress in thelevels of

283

MEP-induced VCAM-1 andICAM-1 expressionin HUVECs. It was

284

foundthat pretreatmentwith

a-tocopherol but not with ascorbic

285 acid or catalase inhibited MEP-induced VCAM-1 and ICAM-1

286

expression (Fig. 4B). Pretreatment with

a

-tocopherol significantly 287 inhibited activationof MEP-induced IjB-a degradation (Fig. 4C).

288

Therefore, antioxidants may have induced the inhibition of

289

MEP-induced VCAM-1 and ICAM-1 expression through the

inhibi-290

tion of NF-jB activation in HUVECs.

Fig. 3. The effects of MEP on NF-jB activation. (A) MEP 30lg/ml treatment induced p65 nuclear localization in a time-dependent manner. TNF-a(T) 10 ng/ml was used as the positive control. (B) MEP treatment induced cytosolic IjB degradation in both dose- and time-dependent manner in HUVECs. (C) NF-jB inhibitor BAY 11-7085 pretreatment inhibited MEP-induced VCAM-1 and ICAM-1 induction. The contents of target protein were analyzed by densitometry. Data were expressed as mean ± S.E.M. and statistical analysis was performed on the results of at least three independent biological replicates.⁄

p < 0.05,⁄⁄

p < 0.01, and⁄⁄⁄

p < 0.001, indicate a statistical difference (n P 3). The control cells were treated with 0.1% DMSO.

C.-C. Lee et al. / Toxicology in Vitro xxx (2012) xxx–xxx 5

291 3.4. Carbon black contributed to MEP-induced VCAM-1 and ICAM-1

292 expression and polycyclicaromatichydrocarbons participatedin

MEP-293 induced ICAM-1 expression

294 We investigated the effects of major compounds in MEP, carbon

295 black (CB), and PAHs on VCAM-1 and ICAM-1 expression for24 hin

296 HUVECs. The results showed that CB nanoparticles with a diameter

297 of15 nm(CB15),29 nm(CB29),51 nm(CB51), and95 nm(CB95)

298 could all induce VCAM-1 protein expression; CB15, CB51, and

299 CB95induced expressionin a dose-dependent manner. Moreover,

300 CB15, CB29, CB51, and CB95 induced ICAM-1 protein expression

301 in a dose-dependent manner. Among thefour typesof carbon black

302 nanoparticles, CB15 induced the highest level of VCAM-1 and

303 ICAM-1 expression (Fig. 5A). Next, among the 8PAHs,

benzo[a]pyr-304 ene (BEN),chrysene (CHY),fluoranthene (FA), phenanthrene (PHE),

305

andpyrene (PYR) inducedincreased ICAM-1 expression in HUVECs,

306 whereas anthracene (ANT), fluorene (FE),and naphthalene (NAP)

307

did not affect ICAM-1expression (Fig. 5B). In addition, we found

308

that none of the 8 PAHs induced VCAM-1 expression (Fig. 5B).

309

4. Discussion

310

Expression of cell adhesion molecules in activated endothelial

311

cells is an initial response in cardiovascular inflammation.

Inte-312

grins expressed on leukocytes bind to ICAM and VCAM and enable

313

leukocytes to adhere to endothelial cells; this is followedby diape-314 desis and induction of tissue inflammation (Wood et al., 1993).

Fig. 4. The effects of antioxidants on MEP-induced VCAM-1 and ICAM-1 induction and IjB degradation in HUVECs. (A) MEP 30lg/ml-induced hydrogen peroxide and superoxide formation. Pretreatment of antioxidants,a-tocopherol (TOC) 30lM, but not by 30lM ascorbic acid (ASC) or 3000 U catalase (Cat), decreased MEP-induced hydrogen peroxide and superoxide production. (B) MEP-induced VCAM-1 and ICAM-1 induction could be significantly inhibited bya-tocopherol (TOC), but not by ascorbic acid or catalase. (C) MEP-mediated IjB degradation could be reduced bya-tocopherol pretreatment. The contents of target protein were analyzed by densitometry. Data were expressed as mean ± S.E.M. and statistical analysis was performed on the results of at least three independent biological replicates.⁄

p < 0.05,⁄⁄

p < 0.01, and⁄⁄⁄

p < 0.001, indicate a statistical difference with control (n P 3).#

p < 0.01, indicate a statistical difference with MEP 30lg/ml treated group. The control cells were treated with 0.1% DMSO.

6 C.-C. Lee et al. / Toxicology in Vitro xxx (2012) xxx–xxx

Please cite this article in press as:Lee, C.-C., et al. Motorcycleexhaust particles up-regulate expressionofvascular adhesion molecule-1 and intercellular adhesion molecule-1inhuman umbilical vein endothelial cells. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2012.01.021

315 This is the first studyto provideexperimental evidence that MEP,

316 the major airborne pollutant in Taiwan, increase ICAM-1 and

317 VCAM-1 expression in HUVECs. To elucidate the mechanisms 318 underlying MEP-induced ICAM-1 and VCAM-1 expression, we

319 demonstrated that MEP induced transcription of ICAM-1 and

320 VCAM-1 and activated NF-jB via IjB-adegradation and p65

trans-321 location to the nucleus (Fig. 6).

322 NF-

j

Bis a major transcription factor involved in the

transcrip-323 tional regulation of cell adhesion molecules and inflammatory

324 genes, and previousstudies have shown that NF-jB is involved

325 in the activation of VCAM-1 and ICAM-1 gene expression (Liao

326 et al., 2008). Oxidativestress has been implicatedin theexpression

327 of ICAM-1 induced bydiesel exhaust particles inhuman bronchial

328 epithelial cells (Takizawa et al., 2000).Further, oxidativestress has

329 also been implicated in NF-

j

B activation by TNF-a stimulation

330 (Rahman et al., 2002).By performingDCFH-DA andDHE staining, 331 we also found that MEPsinduced oxidativestress. MEP-induced

332 expression of VCAM-1 and ICAM-1 inHUVECs was inhibited by

333

a-tocopherol but not by ascorbic acid and catalase.

a

-Tocopherol

334 (Vitamin E) isknown to belong to the most powerful group of

li-335 pid-soluble chain-breakingantioxidants that prevent lipid

peroxi-336

dation and disrupt membrane integrity (Ingold et al., 1987). A 337

previous study also found that

a-tocopherol inhibited the

expres-338

sion of ICAM-1 and VCAM-1 induced by platelet activation

fac-339

tor-1 inHUVECs (Yoshikawa et al., 1998). In this study, we found

340

thatMEP-induced I

j

Bdegradation was inhibited by

a

-tocopherol 341 (Fig. 4C). Therefore, inhibition of MEP-induced expression of

342

VCAM-1 and ICAM-1 by

a

-tocopherol maybe through mitigation

343

of NF-jB activation.

344

Ambient air pollution particles (PM), including diesel exhaust

345

particles, MEP, and those in cigarette smoke, are classified as

346

coarse (aerodynamic diameter, 2.5–10

lm; PM10), fine

(aerody-347

namic diameter, 0.1–2.5

l

m; PM2.5),and ultrafine(aerodynamic 348 diameter, 60.1

l

m).Many studies report that PMsinduce an

in-349

crease in adhesionmolecule expressionin vivo and in vitro (Ando

350

et al., 2001; Delfino et al., 2008; Ishii et al., 2005); however, there

Fig. 5. The effects of different particle sizes of carbon black (CB) and polycyclic aromatic hydrocarbons (PAHs) on VCAM-1 and ICAM-1 induction in HUVECs. (A) CB-induced VCAM-1 and ICAM-1 induction in a dose-dependent manner. (B) Western blot analysis showed the effects of PAHs toward inducing ICAM-1 induction in HUVECs. (ANT, anthracene; BEN, benzo[a]pyrene; CHY, chrysene; FA, fluoranthene; FE, fluorene; NAP, naphthalene; PHE, phenanthrene; PYR, pyrene). The contents of target protein were analyzed by densitometry. Data were expressed as mean ± S.E.M. and statistical analysis was performed on the results of at least three independent biological replicates.

⁄

p < 0.05,⁄⁄

p < 0.01, and⁄⁄⁄

p < 0.001, indicate a statistical difference with control (n P 3). The control cells were treated with 0.1% DMSO.

C.-C. Lee et al. / Toxicology in Vitro xxx (2012) xxx–xxx 7

351 has been noreport to compare the different sizes ofPMs onthe

352 expression of adhesion molecules in HUVECs. In this study, we

353 found that carbon black nanoparticles with different diameters

354 including 15, 29, 51, and 95 nm induced VCAM-1 and ICAM-1

355 expression in a dose-dependent manner. In addition, we found

356 that the smallest carbon black nanoparticles, those with a

diam-357 eter of 15nm, inducedthe highest levels of VCAM-1 and

ICAM-358 1 expression when HUVECs were treated with a low dose

359 (0.05

l

g/ml)of carbon black nanoparticles with different

diame-360 ters. It may indicate that smaller carbon black nanoparticles have

361 a higher potential of inducing arterial inflammation; however,

362 this requires further investigation.

363 Particle-free MEPs are primarily composed of chemicals that

364 include PAHs. Both carcinogenic PAHs (including

benzo[a]anthra-365 cene, chrysene, benzo[a]pyrene, ideno(1,2,3,e,d)pyrene, and

ben-366 zo[g,h,i]pyrene) and noncarcinogenic PAHs (naphthalene,

367 acenaphylene, acenaphene, fluorene, phenathrene, anthracene,

368 and pyrene) have been identified in MEP. In this study, we found

369 that 5-ring PAHs such as benzo[a]pyrene and chrysene induced

370 higher levels of ICAM-1 expression than PAHs with low ring

num-371 bers, such as naphthalene, anthracene, fluorene, and

phenan-372 threne. However, to our surprise, none of the 8 PAHs tested in

373 our study could induce VCAM-1 expression. A previous study

374 found that the signaling pathways of activation of ICAM-1 and

375 VCAM-1 induced byTNF-

a

onHUVECs are different. The

extracel-376 lular signal-regulated kinase (ERK) phosphorylation was involved

377 in TNF-a-induced ICAM-1 expression and PI3K/Akt and protein

378 kinase C (PKC) was involved in TNF-a-induced ICAM-1expression 379 (Hwa et al., 2011; Nizamutdinova et al., 2008). Whether PAHs

in-380 duced ICAM-1 activation through ERK activation but notPI3 K/Akt 381 and PKC pathways on HUVECs needs further investigation.

382 In addition, previous animal studies found that diesel exhaust

383 particles and ultrafine particles induce thrombosis (Nemmar et al.,

384 2003,2002). Further,it iswell known that thrombosis underlies

385 most acute complications of atherosclerosis, such as acute

myocar-386 dialinfarction (Libby, 2001). However, the effects of MEP on

throm-387 bosis requirefurther investigation.

388 In conclusion, this study demonstrated that the MEPmight have 389 adverse effect on human endothelial cells through induction of

390 adhesion molecules-ICAM-1 and VCAM-1 expression in HUVECs.

391

Conflict of Interest Statement

392

We certify that there is no conflict of interest with any

organi-393

zation regarding the material discussed in the manuscript.

394

Acknowledgments

395

This study was supported byGrantsfrom the Taiwan National

396

Science Council (NSC93-2320-B002-118), and China Medical

397

University in Taiwan(CMU99-S-28 andCMU100-S-12).

398

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