chlamydia pneumoniae infection promotes vascular...

Chlamydia pneumoniae infection promotes vascular smooth muscle 1

cell migration through Toll-like receptor 2-related signaling 2

pathway 3

4

Beibei Wang1, Lijun Zhang

1, Tengteng Zhang

1, Haiwei Wang, 5

Junxia Zhang, Junyan Wei, Bingling Shen, Xin Liu, Zhelong Xu, Lijun Zhang# 6

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Department of Pathophysiology, School of Basic Medical Sciences, Tianjin Medical 9

University, Tianjin 300070, China

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#Corresponding author: Lijun Zhang, Department of Pathophysiology, School of Basic 13

Medical Sciences, Tianjin Medical University, No.22 Qixiangtai Road, Heping District, 14

Tianjin 300070, China. Tel: +86 22 8333 6830; Fax: +86 22 8333 6985. E-mail address: 15

[email protected]. 16

1Beibei Wang, Lijun Zhang and Tengteng Zhang contributed equally to this work.

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IAI Accepts, published online ahead of print on 30 September 2013Infect. Immun. doi:10.1128/IAI.01087-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract 22

The migration of vascular smooth muscle cells (VSMCs) from the media to the intima is 23

proposed to be a key event in the development of atherosclerosis. Recently, we reported 24

that Chlamydia pneumoniae infection is involved in VSMC migration. However, the 25

exact mechanisms for C. pneumoniae infection-induced VSMC migration are not yet well 26

elucidated. In this study we examined the role of the Toll-like receptor 2 (TLR2) 27

activation-related signaling pathway in VSMC migration induced by C. pneumoniae 28

infection. An Affymetrix based gene expression array was conducted to identify the 29

changes of gene expression in rat primary VSMCs (rVSMCs) infected with C. 30

pneumoniae. Both the microarray analysis and quantitative real-time RT-PCR revealed 31

that TLR2 mRNA expression was strongly upregulated 12 h after C. pneumoniae 32

infection. RT-PCR and western blot analysis further showed that the expression levels of 33

TLR2 mRNA and protein significant increased at the different time points after infection. 34

Immunocytochemical analysis suggested a TLR2 recruitment to the vicinity of C. 35

pneumoniae inclusions. Cell migration assays showed that the TLR2 neutralizing 36

antibody could significantly inhibit C. pneumoniae infection-induced rVSMC migration. 37

In addition, C. pneumoniae infection stimulated Akt phosphorylation at Ser 473, which 38

was obviously suppressed by the PI3K inhibitor LY294002, thereby inhibiting rVSMC 39

migration caused by C. pneumoniae infection. Furthermore, both the infection-induced 40

Akt phosphorylation and rVSMC migration were suppressed by the TLR2 neutralizing 41

antibody. Taken together, these data suggest that C. pneumoniae infection can promote 42


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VSMC migration possibly through TLR2-related signaling pathway. 43

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Introduction 64

Chlamydia pneumoniae is an obligate intracellular bacterium associated with respiratory 65

tract infection. Moreover, atherosclerosis is a chronic inflammatory disease developing in 66

response to injury in the arterial wall (1), indicating that infectious agents may contribute 67

to atherogenesis. Accumulating evidence indicates that the infection of C. pneumoniae 68

could play a role in the initiation and progression of atherosclerosis (2,3). However, how 69

C. pneumoniae infection contributes to atherosclerosis remains unclear. The migration of 70

vascular smooth muscle cells (VSMCs) from the media to the intima is regarded as a key 71

event in the development of atherosclerosis. Understanding the mechanisms involved in 72

VSMC migration and ultimately the development of strategies by which this process can 73

be inhibited, have been the major focuses of research. 74

Cell migration is believed to be under the control of complex regulatory mechanisms at 75

multiple levels. Recently, C. pneumoniae infection has been shown to be involved in the 76

migration of monocytes (2), HEp-2 cells (4), and VSMCs (5). The exact mechanisms of 77

C. pneumoniae infection-induced VSMC migration have not yet been fully elucidated, 78

although we have reported that C. pneumoniae infection promotes VSMC migration 79

possibly through IQ domain GTPase-activating protein 1 (IQGAP1) (5). Therefore, 80

further understanding of the mechanisms of C. pneumoniae infection-induced VSMC 81

migration may provide important new evidence supporting the pathogenic role of C. 82

pneumoniae in atherosclerosis. 83

Toll-like receptor 2 (TLR2) is a pattern recognition receptor that emerged as a critical 84


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component in the induction of innate immune and inflammatory responses (6,7). TLR2 is 85

expressed in most cardiovascular cells, including endothelial cells (8), VSMCs (9), and 86

macrophages (10), and is thought to be essential in the microbial detection and host cell 87

activation. As a membrane surface receptor, TLR2 recognizes a variety of pathogens, 88

including different bacteria and yeast. Yang et al. found that TLR2 mRNA expression 89

was upregulated when VSMCs were exposed to C. pneumoniae (9). Excitingly, TLR2 has 90

been demonstrated to be able to mediate microvascular endothelial cell migration (11). 91

TLR2 activation could result in the increases in the expressions of intercellular adhesion 92

molecule-1, vascular cell adhesion molecule-1 and chemokines, thereby promoting 93

neutrophil transendothelial migration (11,12). In addition, TLR2 is also thought to have 94

important effects on the starting procedure of the transmigration of polymorphonuclear 95

leucocytes (13). Taken together, these studies indicate a close association of TLR2 with 96

cell migration. 97

Akt, a serine threonine kinase known as protein kinase B, has been shown to play a 98

significant regulatory role in cell migration (14). Akt activation is regulated primarily by 99

phosphorylation at two sites: a conserved threonine residue (Thr 308) by 100

phosphatidylinositol-dependent kinase 1 (PDK1) in the activation loop (15) and a serine 101

residue (Ser 473) by PDK2 in the hydrophobic motif (16). The receptor activator for 102

nuclear factor țB ligand was found to increase the migration of breast cancer cells by 103

activating Akt (17). Lang et al. (18) reported that H2O2 elicited migration of VSMCs by 104

activating Akt signaling pathway. Activation of Akt has been shown in rat (19), human 105


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aortic, and coronary VSMCs (20). Chan et al. (21) found that simvastatin-induced 106

inhibition of VSMC migration involves the suppression of Akt activity. Recent evidence 107

showed that stimulation of TLR2 activates the Akt signaling pathway (22,23). Previous 108

studies demonstrated that C. pneumoniae may stimulate or enhance innate immune and 109

inflammatory response via TLR2, indicating a central role of TLR2 in C. 110

pneumoniae-related cellular signaling (24,25). Therefore, it is possible that activation of 111

TLR2 could activate the Akt signaling pathway during C. pneumoniae infection. Whether 112

TLR2-related Akt signaling pathway mediates C. pneumoniae infection-induced VSMC 113

migration is not well defined. 114

In the present study, we investigated the role of TLR2 in rat primary VSMCs (rVSMCs) 115

migration induced by C. pneumoniae infection and examined the effects of C. 116

pneumoniae infection on Akt activity in rVSMCs, and then explore the role of the 117

activation of TLR2-related signaling pathway in C. pneumoniae infection-induced VSMC 118

migration. 119

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Materials and Methods 121

Antibodies. The following antibodies were used: primary mouse polyclonal anti-C. 122

pneumoniae (CPN0308), which was kindly provided by Dr. Guangming Zhong (San 123

Antonio, USA), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2 neutralizing 124

antibody (AbD Serotec, Kidlington, UK), rabbit anti-Akt and anti-phospho-Akt 125

monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, USA), mouse 126


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anti-ȕ-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., 127

Ltd., Beijing, China). Secondary antibodies included rabbit anti-goat IgG labeled with 128

Cy3 (Bioss, Beijing, China), rabbit anti-mouse IgG labeled with FITC, donkey anti-goat 129

IgG-HRP (Santa Cruz, CA), goat anti-rabbit antibody IgG-HRP and goat anti-mouse 130

IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., PA, USA). 131

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Infection of rVSMCs with C. pneumoniae. C. pneumoniae strain AR-39 (ATCC, 53592) 133

was propagated in HEp-2 cells (ATCC, CCL-23) and purified by gradient centrifugation 134

as described previously (26).

Purified organisms were suspended in 135

sucrose-phosphate-glutamic acid buffer, incubated for 1 h at 4°C and stored at -80°C until 136

use. The infectivity titers were determined by titration in HEp-2 cells monolayers (27). 137

rVSMCs were prepared from the thoracic aorta of 8-week-old male Sprague-Dawley rats 138

by the explant method and cultured in DMEM supplemented with 10% FBS, 25 mg/ml 139

vancomycin and 10 mg/ml gentamicin as described previously (5). rVSMCs were seeded 140

at a density of 3×105 cells/well into 6-well plates. Once confluent, the cells were 141

serum-starved for 12 h to achieve synchronous growth arrest. rVSMCs were then infected 142

with 5×105 inclusion forming units (IFU) (28). The plates were centrifuged at 1700×g for 143

50 min and then incubated at 37°C for 2 h. Subsequently, the medium was replaced with 144

the culture medium with cycloheximide (2 ȝg/ml). 145

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RNA isolation. Total RNA was isolated from C. pneumoniae-infected rVSMCs using 147


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UNlQ-10 column type total RNA extraction kit (Sangon, Shanghai, China) as described 148

by the manufacturer. Degradation of the RNA was excluded by electrophoresis under 149

RNase-free conditions, and the amount was quantified by spectrophotometry using 150

Syngene genesnap (Bio-rad, Hercules, CA). 151

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Affymetrix gene chip. Starting with 10 ȝg of total RNA, samples were labeled and 153

hybridized to the rat expression arrays 230 2.0 (Affymetrix, Santa Clara, CA) according 154

to the manufacturer’s protocol. Microarrays were scanned using the GeneChip® Scanner 155

3000 7G (Affymetrix, Santa Clara, CA) and the raw data was extracted from the scanned 156

images and initially analyzed for general assay quality using Affymetrix Microarray Suite 157

v5.0 software. CEL files were subsequently imported into the program RMAExpress 158

(v0.2 release) for global normalization and generation of expression values. Significance 159

analysis was performed with the significance analysis of microarrays (SAM) algorithm 160

(v1.15). 161

162

Standard and quantitative reverse transcription (RT) PCR. cDNA was prepared from 163

RNA using 200 U of Moloney murine leukemia virus reverse transcriptase (Takara, Otsu, 164

Japan) according to a standard protocol. For standard PCR, the generated cDNA was 165

amplified by PCR using specific primers ᧶ TLR2, 166

5'-GCTCTCCTGTTGTGCTTCTCCAC-3' (forward) and 167

5'-CAGGAGCAGATGAAATGGTTGT-3' (reverse) (29); GAPDH, 5'- 168


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AGTTCAACGGCACAGTCAAG-3' (forward) and 169

5'-TTGTCTGTGGGACACTGCTC-3' (reverse). PCR was started at 95°C for 10 min, and 170

subsequent amplification was performed as follows: for TLR2, 94°C for 15 sec, 58°C for 171

15 sec, and 72°C for 15 sec for 30 cycles; for GAPDH, 94°C for 15 sec, 60°C for 15 sec, 172

and 72°C for 15 sec for 30 cycles and 72°C for 10 min in a microprocessor-driven thermal 173

cycler (Eppendorf, Hannover, Germany). Following agarose gel electrophoresis of the 174

standard PCR products, expression levels were determined semiquantitatively by 175

densitometry. Quantitative real-time RT-PCR was performed with the ABI SDS 7500 176

(Applied Biosystems). 20 µl

reaction containing each primer and 1 µl of 177

reverse-transcribed cDNA was prepared without changing the Mg2+

concentration. PCR 178

conditions were as follows: after an initial denaturation step at 95°C for 30 sec, 40 cycles 179

of denaturation (95°C for 5 sec), annealing, and synthesis (60°C for 34 sec) were 180

performed. The amount of each gene in each sample was relatively quantified using 181

threshold cycle values. 182

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Western blot analysis. Whole cellular protein in rVSMCs was obtained with the RIPA 184

lysis buffer (Beyotime, Haimen, China) for Western blot. The aliquots were separated on 185

SDS-PAGE (10%) and transferred to polyvinylidene fluoride membranes. Then, the 186

membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 187

5% nonfat dry milk and incubated overnight at 4ºC with goat anti-TLR2 polyclonal 188

antibody (1:200 dilution), rabbit anti-Akt monoclonal antibody (1:1000 dilution), rabbit 189


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anti-phospho-Akt (Ser 473) monoclonal antibody (1:1000 dilution) or mouse anti-ȕ-actin 190

monoclonal antibody (1:10000 dilution). After incubated with donkey anti-goat IgG-HRP 191

(1:5000 dilution), goat anti-rabbit IgG-HRP (1:10000 dilution) or goat anti-mouse 192

IgG-HRP (1:10000 dilution), proteins were detected by an enhanced chemiluminescence 193

kit (Beyotime, Haimen, China). The Western blot bands were scanned and 194

semi-quantified by densitometry. 195

196

Confocal microscopy. rVSMCs were plated onto 12-mm circular coverslips in 24-well 197

plates (1.5×104 cells/well) and cultured overnight. After C. pneumoniae infection, cells 198

were fixed with methanol and permeabilized with 0.1% Triton X-100, then incubated for 199

3 h at 4°C with blocking buffer containing antibody against C. pneumoniae and antibody 200

against TLR2. After washing with phosphate buffer solution, cells were incubated with 201

the corresponding secondary antibodies conjugated with FITC or Cy-3. Coverslips were 202

washed three times, mounted with Prolong Antifade (Molecular Probes, Pitchford, OR) 203

and visualized on an LSM 510 confocal laser scanning microscope (Zeiss, Thornwood, 204

NY) using LSM 510 software for image analysis. 205

206

Cell migration assays. Wound-healing and Transwell migration assays were performed 207

as previously described (5) with some modifications. Confluent cells were treated with 208

serum-free medium containing hydroxyurea (Sigma, St. Louis, USA) for 12 h before 209

starting the experiments (30). After incubation with hydroxyurea, 10 µg/ml of TLR2 210


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neutralizing antibody (31) or 25 ȝM of the PI3K inhibitor LY294002 (Promega, Madison, 211

USA) was added to the cells for 1 h. 212

For the wound-healing assay, a single uniform scratch was carefully made using a sterile 213

200 ȝl yellow plastic pipette tip. After carefully washed to remove the detached cells, 214

cells were infected with C. pneumoniae (at an infectious dose of 5×105 IFU). Multiple 215

photographs of the wounds were then taken at 0 h and 24 h post-wounding under an 216

inverted Nikon microscope (Nikon Corporation, Tokyo, Japan) at 100× magnification. 217

The areas of cell recoverage were determined with image analysis software (ImageJ). The 218

ratio of cellular recoverage area to the whole wound area was used to evaluate migration. 219

For the Transwell migration assay, rVSMCs were infected with C. pneumoniae for 14 h. 220

Then, cells were seeded into the upper chamber of the transwell inserts (1.5×104 cells per 221

well) (Corning, St. Lowell, MA). Cells were subsequently allowed to migrate for 8 h at 222

37°C. The cells on the upper side of the inserts were softly scraped off. The cells that 223

migrated to the lower side of the inserts were fixed with 4% paraformaldehyde and 224

stained with 0.1% crystallin violet, and then the cells from nine independent, randomly 225

chosen visual fields were counted in an inverted phase contrast microscope (200× 226

magnification) for quantification of cells. 227

228

Statistical analysis. All experiments were repeated in triplicate, and all statistical 229

analyses were performed using SPSS 16.0. Student’s t-test was used for the comparisons 230

between two different groups. To compare between multiple experimental groups, 231


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one-way ANOVA followed by the Tukey’s post hoc analysis was performed. The results 232

were considered statistically significant at P < 0.05. 233

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Results 235

C. pneumoniae stimulates TLR2 expression in rVSMCs. To exlpore whether C. 236

pneumoniae infection could upregulate TLR2 mRNA expression in rVSMCs, microarray 237

was performed to assess the gene expression in rVSMCs 12 h after infection. Of the 238

31,042 transcripts (28,000 well-characterized genes) presented on the chips, 35 genes 239

were found to be differentially regulated by C. pneumoniae infection and satisfied the 240

comparison filtering criteria. Among them, 23 genes were downregulated and 12 were 241

upregulated, including the TLR2 gene (2.21 fold-change) (Table). The change of TLR2 242

mRNA expression was confirmed by TaqMan quantitative real-time RT-PCR (Fig. 1A) 243

using the same RNA samples that had been used for the microarray assay in the same 244

manner and at the same time points. The changes in the expression levels of the validated 245

genes showed the similar patterns in microarray and real-time RT-PCR 12 h after the 246

infection. 247

To further examine the effects of C. pneumoniae infection on TLR2 expression, the 248

standard PCR was performed to determine the expression levels of TLR2 mRNA at 249

different time points in the infected rVSMCs. TLR2 expression was detected 0 h, 2 h, 4 h, 250

6 h, 8 h, 10 h, 12 h, and 24 h after C. pneumoniae infection. The amount of TLR2 mRNA 251

2 h after infection roughly doubled compared to the control group, and reached the peak 252


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at 8 h postinfection with a prolonged expression until 24 h after C. pneumoniae infection 253

(Fig. 1B). To confirm this finding, TLR2 protein levels were determined with western 254

blot in C. pneumoniae infected rVSMCs. As shown in Fig. 1C, the expression levels of 255

TLR2 protein were increased significantly 12 h and 24 h after C. pneumoniae infection. 256

TLR2 protein expression at 12 h postinfection was higher than one seen 24 h after 257

infection, there was no significant difference between the two groups. 258

259

C. pneumoniae-infected rVSMCs recruit TLR2 around the inclusions. To analyze the 260

subcellular localization of TLR2 in rVSMCs infected with C. pneumoniae, the 261

immunocytochemistry analysis was performed. Under fluorescence confocal microscopy, 262

a scattered cytoplasmic distribution of TLR2 without a specific pattern was apparent in 263

noninfected rVSMCs. In contrast, in C. pneumoniae-infected rVSMCs, large cytoplasmic 264

inclusions were observed, and the expression of TLR2 was increased, especially around 265

the C. pneumoniae inclusions (Fig. 2). 266

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TLR2 mediates C. pneumoniae infection-induced rVSMC migration. Cell migration 268

assays were performed to assess whether TLR2 was involved in C. pneumoniae 269

infection-induced rVSMC migration. In the wound healing assay, C. pneumoniae 270

infection accelerated the rVSMC migration velocity compared with the control group 24 271

h after the cell monolayer was scratched with a pipette tip (0.913 ± 0.031 vs. 0.623 ± 272

0.040, P < 0.05). Compared with C. pneumoniae-infected rVSMCs, the cell migration 273


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velocity was significantly reduced after the use of a TLR2 neutralizing antibody (10 274

µg/ml) (0.913 ± 0.031 vs. 0.763 ± 0.057, P < 0.05, Fig. 3A). In addition, the Transwell 275

migration assay showed that the number of rVSMCs infected with C. pneumoniae 276

migrated through the membrane was more than the control group (55.32 ± 7.72 vs. 33.92 277

± 5.96, P < 0.05). The TLR2 neutralizing antibody markedly suppressed C. pneumoniae 278

infection-induced rVSMC migration compared with C. pneumoniae infection group 279

(36.95 ± 6.27 vs. 55.32 ± 7.72, P < 0.05, Fig. 3B). 280

281

C. pneumoniae infection induces Akt phosphorylation in rVSMCs. Phosphorylation of 282

Akt is critical for its effects on cell migration. Accordingly, we determined whether C. 283

pneumoniae infection induces Akt phosphorylation in rVSMCs. As shown in Fig. 4, there 284

was no significant difference in levels of total Akt protein expression between the control 285

and C. pneumoniae-infected rVSMCs, whereas the level of Akt phosphorylation at Ser 286

473 was increased significantly at different time points after C. pneumoniae infection. 287

The amount of phosphorylated Akt began to increase at 0.5 h after infection, and reached 288

the peak at 1 h postinfection with a prolonged high level of Akt phosphorylation and then 289

decreased at 12 h and 24 h after infection (Fig. 4). 290

291

Suppression of Akt phosphorylation inhibits C. pneumoniae infection-induced 292

rVSMC migration. To further address the significance of Akt phosphorylation in C. 293

pneumoniae infection-induced rVSMC migration, we blocked the phosphorylation of Akt 294


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by the use of the PI3K inhibitor LY294002 (32) (1.50 ± 0.123 vs. 0.74 ± 0.009, P < 0.05, 295

Fig. 5A), and then the changes in cell migration were observed in the in vitro model of 296

rVSMC monolayer wounding and restitution. The results showed that exposure to C. 297

pneumoniae contributed to the acceleration of the wound closure (0.783 ± 0.034 vs. 0.557 298

± 0.062, P < 0.05). Furthermore, the infection-induced rVSMC migration was markedly 299

inhibited after the suppression of Akt phosphorylation by LY294002 when compared with 300

the cells only infected with C. pneumoniae (0.562 ± 0.052 vs. 0.783 ± 0.034, P < 0.05, 301

Fig. 5B). 302

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TLR2 neutralizing antibody suppresses Akt phosphorylation induced by C. 304

pneumoniae infection. To analyze whether TLR2 mediates the promoting effect of C. 305

pneumoniae infection on VSMC migration by activating Akt, we blocked TLR2 function 306

using the TLR2 neutralizing antibody to determine the involvement of Akt in TLR2 307

signaling pathway during C. pneumoniae infection. Western blot analysis showed that the 308

elevated Akt phosphorylation level induced by C. pneumoniae infection was suppressed 309

by the TLR2 neutralizing antibody (2.03 ± 0.251 vs. 2.96 ± 0.15, P < 0.05, Fig. 6). 310

311

Discussion 312

Despite the close link between C. pneumoniae infection and atherosclerosis (2,5), the 313

exact mechanisms by which C. pneumoniae infection may lead to the development of 314

atherosclerosis is still not well understood. VSMC migration is known to be an essential 315


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element in the development of atherosclerotic lesions. Previous studies have found that C. 316

pneumoniae infection can indirectly induce the transendothelial migration of neutrophils 317

and monocytes by stimulating human coronary artery endothelial cells to secrete 318

chemokines and adhesion molecules (33,34). Recently, Schmidt et al. found that C. 319

pneumoniae infection can directly induce monocyte migration through Matrigel (2). In 320

addition, we have reported previously that C. pneumoniae infection can also directly 321

promote the migration of HEp-2 cells (4) and VSMCs (5). Our previous study revealed 322

that C. pneumoniae infection-induced VSMC migration may attribute to the upregulation 323

of IQGAP1 expression (5). However, how C. pneumoniae infection regulates IQGAP1 324

remains unknown. Since IQGAP1 is only one of the cytoskeletal regulatory proteins, it is 325

reasonable to hypothesize that C. pneumoniae interacts with more than one of the key 326

intracellular proteins involved in cell migration, especially the proteins required for 327

recognizing C. pneumoniae and transmitting signals. 328

As a possible pattern recognition receptor for C. pneumoniae, TLR2 has also been shown 329

to affect cell migration (13). However, whether TLR2 participates in VSMC migration 330

caused by C. pneumonia infection is still unclear. To explore the potential mechanism 331

involved in the infection-induced VSMC migration, microarray assay, RT-PCR and 332

western blot were performed to observe the effects of C. pneumonia infection on TLR2 333

mRNA and protein expressions. Our data showed that TLR2 mRNA expression was 334

markedly upregulated in C. pneumoniae infected rVSMCs compared with the control, 335

which is consistent with the results reported by Yang et al. (9) who found that 336


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intracellular TLR2 may be responsible for the initiation of signal transduction events 337

during C. pneumoniae infection. The expression of TLR2 mRNA began to increase 338

within 2 h after C. pneumoniae infection with a maximum 8 h after the infection and 339

sustained for 24 h after the infection. Likewise, TLR2 protein expression levels were 340

found to be significantly upregulated 12 h and 24 h after the infection, suggesting that the 341

rapid effects of C. pneumoniae exposure and the persistent responses to C. pneumoniae 342

infection in rVSMCs are both mediated by TLR2. These results also indicate that the 343

expression of TLR2 in rVSMCs may mediate cellular responses to C. pneumoniae 344

components at a post-transcriptional level. 345

We found that C. pneumoniae infection not only upregulated the expression level of 346

TLR2, but also changed the TLR2 subcellular localization by promoting translocation of 347

TLR2 to the vicinity of the inclusions. Similar results were observed in prostate epithelial 348

cells infected by Chlamydia muridarum (35). Previous studies revealed that C. 349

pneumoniae is taken up into cellular hosts within 2 h after cells were exposed to the 350

elementary bodies (36). The cytokines are also released from the infected cells in a 351

TLR2-dependent manner at 24 h postinfection (25,37). Our data showed that the infected 352

rVSMCs actively recruited TLR2 around the intracellular chlamydial inclusion, implying 353

that TLR2 can recognize C. pneumoniae and is responsible for the initiation of signal 354

transduction events upon infection with C. pneumoniae. 355

Recently, much interest has focused on the adaptive role of TLR2 as a key mediator of 356

the innate immune response, inducing synthesis of antimicrobial peptides and cytokines 357


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that promote adaptive immunity. In fact, TLR2 activation may not only induce 358

inflammatory responses that lead to pathogenic conditions, but also be associated with 359

cell migration (11,38). Accordingly, we investigated the role of TLR2 in C. pneumoniae 360

infection-induced rVSMC migration. In this study, we found that C. pneumoniae 361

infection may promote VSMC migration by upregulating the expression of TLR2. In 362

addition, the suppression of TLR2 activity with the TLR2 neutralizing antibody 363

significantly inhibited cell migration induced by C. pneumoniae infection, suggesting a 364

critical role of TLR2 activation in C. pneumoniae infection-induced VSMC migration. 365

Others have also reported that TLR2 signaling in the airway may initiate cleavage of 366

transmembrane junctional proteins to accommodate transmigration of recruited 367

polymorphonuclear leukocytes (13). Moreover, TLR2 was shown to play an important 368

role in the migration of cancer cells as well by stimulating the production of 369

mitogen-activated protein kinase and inhibitor of kappaB kinase, and the blocking of 370

TLR2 activity can attenuate metastasis of the tumor (39,40). The activated TLR2 can 371

stimulate cell-migration not only indirectly by inducing proinflammatory cytokine 372

expression (11,12), but also directly by activating the intracellular dynamical system 373

(41,42). Activation of TLR2 may initiate the TLR2-MyD88-Rac1 signaling pathway 374

which accounts for the molecular mechanism of oxidative stress-induced migration of 375

endothelial cells (39). Lasunskaia et al. (42) demonstrated that morphological cell 376

rearrangements leading to the spreading and polarization of macrophages known to be 377

important for cell migration were mediated by TLR2-dependent PI3K activation. Our 378


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previous study suggested a positive role of IQGAP1 in the regulation of VSMC migration 379

induced by C. pneumoniae infection (5). How TLR2 transmits the signals from the 380

membrane receptor to the cell migration-related downstream effectors after C. 381

pneumoniae infection needs further studies. 382

Akt is known as a key regulator for many signal transduction pathways, modulating 383

multiple cellular functions including cell migration (43,44). However, little is known 384

about the role of Akt activation in the promotive effect of C. pneumoniae infection on the 385

migration of VSMCs. In this study, we found that C. pneumoniae infection induced Akt 386

phosphorylation at Ser 473 in rVSMCs at different time points after infection. Coombes 387

et al. (36) found that Akt phosphorylation at Ser 473 was also detected for the 388

duration of the experiment (2 h) after the addition of C. pneumoniae to the cells. 389

Lately, C. pneumoniae infection has been shown to induce Akt phosphorylations at Thr 390

308 and Ser 473 (45). These studies suggest that C. pneumoniae infection can increase 391

the level of phosphorylated Akt, partially supporting our findings. In contrast, Lin et al. 392

(46) demonstrated that after 6 h of incubation with GroEL1, a heat shock protein 60 of C. 393

pneumoniae, Akt phosphorylation was significantly decreased in human coronary artery 394

endothelial cells. Additional studies are needed to explain these contradictory data about 395

the effects of C. pneumoniae infection on Akt phosphorylation. 396

To further determine the role of Akt phosphorylation in VSMC migration induced by C. 397

pneumoniae infection, we first used the PI3K inhibitor LY294002 to suppress Akt 398

phosphorylation (32), and then investigated the changes in the migration of rVSMCs by 399


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the wound healing assay. Our data showed that C. pneumoniae infection-induced rVSMC 400

migration was significantly inhibited by the pretreatment of rVSMCs with LY294002, 401

indicating that Akt phosphorylation plays an important role in VSMC migration 402

stimulated by C. pneumoniae infection. LY294002 can also inhibit fibroblast growth 403

factor 2-stimulated migration of the periodontal ligament cells through suppressing Akt 404

activation (47). Recently, Walker et al. (48) reported that transforming growth factor-ȕ3 405

significantly induced motility of human prostate cancer PC3 cells by increasing the level 406

of phosphorylated Akt (Ser 473), and LY294002 blocked transforming growth 407

factor-ȕ3-stimulated migration via the suppression of Akt phosphorylation at Ser 473. 408

These studies revealed that Akt phosphorylation is closely associated with cell migration, 409

supporting our hypothesis that C. pneumoniae infection promotes VSMC migration 410

possibly by inducing Akt phosphorylation. 411

Akt pathway has been shown to be involved in TLR2 signaling (22,23). Whether Akt 412

activation participates in TLR2-mediated promoting effects of C. pneumoniae infection 413

on VSMC migration remains unknown. Therefore, we blocked TLR2 function using the 414

TLR2 neutralizing antibody to determine the involvement of Akt in TLR2 signaling 415

pathway during C. pneumoniae infection. Excitingly, we found that Akt phosphorylation 416

induced by C. pneumoniae infection was suppressed by the TLR2 neutralizing antibody, 417

suggesting a partial dependence of Akt activation on TLR2. In the present study, we first 418

demonstrated that C. pneumoniae infection-induced Akt phosphorylation was partially 419

regulated by TLR2 although Akt activation has been shown to be involved in TLR2 420


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signaling pathway in Listeria monocytogenes (49) and mycobacteria (43). We have 421

reported that C. pneumoniae infection may promote the migration of VSMCs by 422

upregulating the IQGAP1 expression (5). Chen et al. (50) found that IQGAP1 can 423

promote cell proliferation by Akt activation, suggesting that IQGAP1 is closely 424

associated with Akt activation. Whether C. pneumoniae infection promotes VSMC 425

migration through IQGAP1 activated by TLR2-related Akt signaling pathway remains to 426

be addressed. 427

In conclusion, we have demonstrated that C. pneumoniae infection can promote rVSMC 428

migration possibly through TLR2-related signaling pathway. Our data suggest the 429

positive roles of TLR2 and Akt phosphorylation in the regulation of C. pneumoniae 430

infection-induced VSMC migration, providing new evidence to support the hypothesis 431

that C. pneumoniae infection accelerates the development of atherosclerosis by 432

stimulating VSMC migration from the media to the intima possibly through activating 433

TLR2-related signaling pathway. 434

435

Conflict of interest 436

The authors have no conflicts of interest. 437

438

Acknowledgments 439

This work was supported by grants (No. 30971225 to Z.L.) from the National Natural 440

Science Foundation of China, the Specialized Research Fund for the Doctoral Program of 441


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Higher Education of China (No. 20111202110011 to Z.L.) and the Key Project of Science 442

Technology (No. 206008 to Z.L.) from China Education Ministry. 443

The authors deeply appreciate the help from Foreign Expert Linda Perkins at Tianjin 444

Medical University for critical reading of the manuscript, and Prof. Guangming Zhong 445

(Department of Microbiology and Immunology, University of Texas Health Science 446

Center at San Antonio, San Antonio, USA) for his gift of the C. pneumoniae polyclonal 447

antibody. 448

449

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622

Figure legends 623

Fig. 1. C. pneumoniae stimulates TLR2 expression in rVSMCs. (A) Validation of TLR2 624

gene expression profiling using quantitative real-time RT-PCR normalized to GAPDH 625

expression. Data shown are mean values of PCR replicates from individual group. (B) 626

TLR2 mRNA expression at the indicated time points after C. pneumonia infection. 627

rVSMCs infected with C. pneumoniae (5×105 IFU) for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h 628


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or control cells were lysed to prepare the total RNA. cDNA was amplified for 30 cycles 629

and PCR products were separated by agarose gel electrophoresis. Relative TLR2 mRNA 630

expression levels determined by standard PCR (n=3 replicates per group). (C) C. 631

pneumoniae infection stimulates TLR2 protein expression in rVSMCs. Cells were 632

infected with C. pneumoniae for 0 h, 12 h or 24 h. Cell lysates were separated by 633

SDS-PAGE and blots were probed with anti-TLR2 and anti-ȕ-actin antibodies, followed 634

by donkey anti-goat IgG-HRP and goat anti-mouse IgG-HRP antibodies, and developed 635

with ECL. 636

637

Fig. 2. Confocal microscopy analysis of TLR2 expression pattern in C. pneumoniae- 638

infected rVSMCs. rVSMCs were grown in coverslips and infected with C. pneumoniae 639

for 60 h, and then were fixed, permeabilized, and stained using specific antibodies. C. 640

pneumoniae inclusions were observed using a mouse polyclonal antibody to C. 641

pneumoniae. Fluorescence micrographs were stained with TLR2-specific antibody. Cell 642

nuclear was stained with DAPI. 643

644

Fig. 3. Effects of TLR2 on rVSMC migration induced by C. pneumoniae infection. The 645

TLR2 neutralizing antibody was added 1 h before C. pneumoniae infection. (A) Wound 646

healing assay. “Scratch-wounds” were created by scraping the confluent cell monolayer 647

with a sterile pipette tip, and then cells were infected with C. pneumoniae at an infectious 648

dose of 5×105 IFU. Photographs were taken of the same wounded area of each well at 0 h 649


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and 24 h. The scratched regions were photographed under an inverted Nikon microscope 650

(100× magnification) at 24 h after C. pneumoniae infection. Migration velocity is 651

presented as a ratio of of cellular recoverage area to the whole wound area. *P < 0.05 vs. 652

control; **

P < 0.05 vs. C. pneumoniae infection group. (B) Transwell migration assay. 653

Cell morphology was observed by staining with 0.1% crystallin violet. The number of 654

cells that had migrated through the pores was quantified by counting nine independent 655

visual fields using a microscope (200× magnification). *P < 0.05 vs. control;

** P < 0.05 656

vs. C. pneumoniae infection group. 657

658

Fig. 4. C. pneumoniae infection induces Akt phosphorylation in rVSMCs. Cells were 659

infected with C. pneumoniae (5×105 IFU) for 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 6 h, 12 h, 24 h. 660

Equal amounts of protein lysates were subjected to SDS-PAGE, and blots were probed 661

with anti-Akt, anti-phospho-Akt (Ser 473) and anti-ȕ-actin antibodies, followed by goat 662

anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP antibodies, and developed with ECL. 663

P-Akt indicates phosphorylated Akt. 664

665

Fig. 5. Suppression of Akt phosphorylation inhibits C. pneumoniae infection-induced 666

rVSMC migration. (A) LY294002 (a phosphoinositide 3-kinase inhibitor) inhibits Akt 667

phosphorylation induced by C. pneumoniae infection. After pretreatment with or without 668

LY294002 (25 ȝM) at 37 ºC for 1 h, rVSMCs were inoculated with C. pneumoniae 669

(5×105 IFU) and incubated for 2 h. Equal amounts of protein lysates were subjected to 670


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SDS-PAGE, and blots were probed with anti-Akt, anti-phospho-Akt (Ser 473) and 671

anti-ȕ-actin antibodies, followed by the corresponding HRP-conjugated secondary 672

antibodies, and developed with ECL. P-Akt indicates phosphorylated Akt. (B) Effects of 673

Akt phosphorylation on C. pneumoniae infection-induced rVSMC migration. After 674

pretreatment with or without LY294002 (25 ȝM) at 37 ºC for 1 h, rVSMCs were 675

inoculated with C. pneumoniae at an infectious dose of 5×105 IFU and incubated for 14 h. 676

Cells were then incubated at 37 ºC for 10 h to allow cell to migrate through the membrane. 677

Migration velocity is presented as a ratio of of cellular recoverage area to the whole 678

wound area. *P < 0.05 vs. control;

** P < 0.05 vs. C. pneumoniae infection group. 679

680

Fig. 6. The TLR2 neutralizing antibody suppresses Akt phosphorylation induced by C. 681

pneumoniae infection. rVSMCs cultured for 24 h in 6-well plates were incubated with the 682

TLR2 neutralizing antibody (10 µg/ml) and then infected with C. pneumoniae. Equal 683

amounts of protein lysates were subjected to SDS-PAGE, and blots were probed with 684

anti-Akt, anti-phospho-Akt (Ser 473) and anti-ȕ-actin antibodies, followed by the 685

corresponding horseradish peroxidase-conjugated secondary antibodies, and developed 686

with ECL. P-Akt indicates phosphorylated Akt. *P < 0.05 vs. control;

** P < 0.05 vs. C. 687

pneumoniae infection group. 688


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1

Fig. 1. C. pneumoniae stimulates TLR2 expression in rVSMCs. (A) Validation of2

TLR2 gene expression profiling using quantitative real-time RT-PCR normalized to3

GAPDH expression. Data shown are mean values of PCR replicates from individual4

group. (B) TLR2 mRNA expression at the indicated time points after C. pneumonia5

infection. rVSMCs infected with C. pneumoniae (5×105 IFU) for 2 h, 4 h, 6 h, 8 h, 106

h, 12 h, 24 h or control cells were lysed to prepare the total RNA. cDNA was7

amplified for 30 cycles and PCR products were separated by agarose gel8

electrophoresis. Relative TLR2 mRNA expression levels determined by standard PCR9

(n=3 replicates per group). (C) C. pneumoniae infection stimulates TLR2 protein10

expression in rVSMCs. Cells were infected with C. pneumoniae for 0 h, 12 h or 24 h.11

Cell lysates were separated by SDS-PAGE and blots were probed with anti-TLR2 and12

anti-β-actin antibodies, followed by donkey anti-goat IgG-HRP and goat anti-mouse13

IgG-HRP antibodies, and developed with ECL.14


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1

Fig. 2. Confocal microscopy analysis of TLR2 expression pattern inC. pneumoniae-2

infected rVSMCs. rVSMCs were grown in coverslips and infected withC. pneumoniae3

for 60 h, and then were fixed, permeabilized, and stained using specific antibodies.C.4

pneumoniaeinclusions were observed using amouse polyclonal antibody toC.5

pneumoniae. Fluorescence micrographs were stained with TLR2-specific antibody. Cell6

nuclear was stained with DAPI.7


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1

Fig. 3. Effects of TLR2 on rVSMC migration induced byC. pneumoniaeinfection. The2

TLR2 neutralizing antibody was added 1 h beforeC. pneumoniaeinfection. (A) Wound3

healing assay. “Scratch-wounds” were created by scraping the confluent cell monolayer4

with a sterile pipette tip, and then cells were infected withC. pneumoniaeat an infectious5

dose of5×105 IFU. Photographs were taken of the same wounded area of each well at 0 h6

and 24 h. The scratched regions were photographed under an inverted Nikon microscope7

(100× magnification) at 24 h afterC. pneumoniaeinfection. Migration velocity is8

presented as a ratio of of cellular recoverage area to the whole wound area.*P < 0.05vs.9

control; ** P < 0.05 vs. C. pneumoniaeinfection group.(B) Transwell migration assay.10

Cell morphology was observed by staining with 0.1% crystallinviolet. The number of11

cells that had migrated through the pores was quantified by counting nine independent12

visual fields using a microscope (200× magnification).*P < 0.05vs.control; ** P < 0.0513

vs. C. pneumoniaeinfection group.14


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1

Fig. 4. C. pneumoniaeinfection induces Akt phosphorylation inrVSMCs. Cells were2

infected withC. pneumoniae(5×105 IFU) for 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 6 h, 12 h, 24 h.3

Equalamounts of protein lysates were subjected to SDS-PAGE, and blots were probed4

with anti-Akt, anti-phospho-Akt (Ser 473)and anti-く-actin antibodies, followed bygoat5

anti-rabbit IgG-HRP andgoat anti-mouseIgG-HRPantibodies, and developed with ECL.6

P-Akt indicatesphosphorylated Akt.7


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1

Fig. 5. Suppression of Akt phosphorylation inhibitsC. pneumoniaeinfection-induced2

rVSMC migration. (A) LY294002 (aphosphoinositide 3-kinaseinhibitor) inhibits Akt3

phosphorylationinduced byC. pneumoniaeinfection.After pretreatmentwith or without4

LY294002 (25たM) at 37 ºC for 1 h, rVSMCs were inoculated withC. pneumoniae5

(5×105 IFU) and incubated for 2 h.Equal amounts of protein lysates were subjected to6

SDS-PAGE, and blotswere probed with anti-Akt, anti-phospho-Akt (Ser 473) and7

anti-く-actin antibodies, followed by the correspondingHRP-conjugated secondary8

antibodies, and developed with ECL.P-Akt indicatesphosphorylated Akt.(B) Effects of9

Akt phosphorylation onC. pneumoniae infection-induced rVSMC migration.After10

pretreatmentwith or without LY294002 (25 たM) at 37 ºC for 1 h, rVSMCs were11

inoculated withC. pneumoniaeat an infectious dose of5×105 IFU and incubated for 14 h.12


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Cells were then incubated at 37ºC for 10 h to allow cell to migrate through the13

membrane.Migration velocity is presented as a ratio of of cellular recoverage area to the14

whole wound area.*P < 0.05vs.control; ** P < 0.05vs. C. pneumoniaeinfection group.15


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1

Fig. 6. The TLR2 neutralizing antibody suppressesAkt phosphorylationinduced byC.2

pneumoniaeinfection.rVSMCscultured for 24 h in 6-well plates were incubated with the3

TLR2 neutralizing antibody (10 µg/ml) and then infected withC. pneumoniae. Equal4

amounts of protein lysates were subjected to SDS-PAGE, and blots were probed with5

anti-Akt, anti-phospho-Akt (Ser 473) and anti-く-actin antibodies, followed by the6

correspondinghorseradish peroxidase-conjugatedsecondary antibodies, and developed7

with ECL. P-Akt indicatesphosphorylated Akt.*P < 0.05vs. control; ** P < 0.05vs. C.8

pneumoniaeinfection group.9


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Table Fold changes in microarray for all 35 geneswere found to be differentially1

regulated by C. pneumoniae infection and satisfied the comparison filtering criteria2

Gene Title Gene SymbolNCBI RefSeq

accession no.

fold

change

proliferating cell nuclear antigen Pcna NM_022381 2.34

transforming growth factor, beta 3 Tgfb3 NM_013174 -3.29

chemokine (C-X3-C motif) ligand 1 Cx3cl1 NM_134455 4.38

early growth response 1 Egr1 NM_012551 -3.51

nuclear factor, interleukin 3

regulatedNfil3 NM_053727 -2.05

A kinase (PRKA) anchor protein 12 Akap12 BG663107 -3.34

inhibitor of DNA binding 2 Id2 NM_013060 5.45

coagulation factor III

(thromboplastin, tissue factor)F3 NM_013057 -4.59

endothelin 1 Edn1 NM_012548 -2.64

chemokine (C-C motif) ligand 20 Ccl20 AF053312 2.45

stathmin-like 2 Stmn2 BG668493 -2.25

minichromosome maintenance

complex component 6Mcm6 U17565 2.35

protein kinase inhibitor, gamma Pkig AA943815 -2.34

phosphatidic acid phosphatase type

2BPpap2b AI177031 -2.56


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phosphoserine aminotransferase 1 Psat1 AI230228 -2.16

ERBB receptor feedback inhibitor 1 Errfi1 AI169756 -2.38

gap junction protein, beta 2 Gjb2 AI179953 -6.79

BAI1-associated protein 2 Baiap2 BI279562 2.14

FBJ osteosarcoma oncogene Fos BF415939 -20.99

--- --- AI008792 4.68

thrombomodulin Thbd AA818521 -2.74

similar to RIKEN cDNA 6330406I15 RGD1307396 AW916327 -2.26

RAS-like family 11 member B Rasl11b BG375198 -2.9

inhibitor of DNA binding 1 Id1 M86708 2.3

transforming growth factor alpha Tgfa NM_012671 -2.2

NAD(P)H dehydrogenase, quinone 1 Nqo1 J02679 2.6

Cd55 molecule Cd55 AB032395 -5.7

similar to limb-bud and heart LOC683626 AA800701 -3.51

minichromosome maintenance

deficient 7 (S. cerevisiae)Mcm7 AI599678 2.17

gremlin 2, cysteine knot superfamily,

homolog (Xenopus laevis)Grem2 AA817956 2.57

transforming growth factor alpha Tgfa BG670310 -2.71

toll-like receptor 2 Tlr2 AW526982 2.21

--- Rn.23094.2 AI548994 -3.32

annexin A1 Anxa1 AI236455 -2.46


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dual specificity phosphatase 1 Dusp1: U02553 #VALUE!

3


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chlamydia pneumoniae infection promotes vascular...

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