archives of oral biology 59 (2014) 944–953

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Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases-1, -2 and -9 during periapical lesion development Nata´lia Guimara˜es Kalatzis Sousa a, Cristina Ribeiro de Barros Cardoso b, Joa˜o Satana da Silva c, Milton Carlos Kuga a, Ma´rio Tanomaru-Filho a, Gisele Faria a,* a Department of Restorative Dentistry, Araraquara Dental School, UNESP—Univ. Estadual Paulista, Rua Humaita´, 1680, 14801-903 Araraquara, SP, Brazil b Department of Clinical Analysis, Toxicology and Food Sciences, Faculty of Pharmaceutical Sciences, University of Sa˜o Paulo, Av. do Cafe´, s/n Monte Alegre, 14040-904 Ribeira˜o Preto, SP, Brazil c Department of Biochemistry and Immunology, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Av. Bandeirantes, 3900, Campus Universita´rio, 14049-900 Ribeira˜o Preto, SP, Brazil

article info

abstract

Article history:

Objective: To evaluate the expression of matrix metalloproteinase inducer (EMMPRIN) and

Received 26 November 2013

its correlation with the expression of matrix metalloproteinases (MMPs)-1, -2 and -9 during

Received in revised form

the development of periapical lesion in mice.

17 May 2014

Methods: Periapical lesions were induced in the lower first molars of mice and after 7, 14, 21

Accepted 21 May 2014

and 42 days the mandibles were removed. The periapical lesions were measured by micro-

Available online

computed tomography. The expression of EMMPRIN, MMPs-1, -2, and -9 genes were determined by real-time RT-PCR. The location and expression of EMMPRIN and MMPs were

Keywords:

evaluated by immunohistochemistry.

Endodontics

Results: At 14 days, the periapical lesion area was higher than at 7 days. At 21 and 42 days no

Periapical lesion

statistically significant bone loss was observed in comparison to 14 days. The control group

Matrix metalloproteinases

showed discrete and occasional EMMPRIM, MMP-1, -2 and -9 immunostaining in the

EMMPRIN

periodontal ligament fibroblasts. At 7, 14, 21 and 42 days intense immunoexpression was observed for EMMPRIN, MMPs-1, -2 and -9 in the region adjacent to the apical foramen. The EMMPRIN immunoexpression was higher at 7, 14, 21 and 42 days compared with the control. There was a positive correlation between gene expression of EMMPRIN and MMPs in the active phase of periapical lesion development. Conclusion: There is a high expression of EMMPRIM mainly by the inflammatory infiltrate in the region adjacent to the apical foramen during periapical lesion development. Furthermore, the positive correlation with MMP-1, -2, and -9 during the first days after periapical lesion induction indicates that EMMPRIM may be involved in the active phase of periapical lesions development. # 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +55 16 3301 6398; fax: +55 16 3301 6392. E-mail address: [email protected] (G. Faria). http://dx.doi.org/10.1016/j.archoralbio.2014.05.021 0003–9969/# 2014 Elsevier Ltd. All rights reserved.

archives of oral biology 59 (2014) 944–953

1.

Introduction

Periapical lesion is caused by the interaction of microbial factors and host defence at the interface between the root canal and periodontal ligament.1 This interaction results in the production of cytokines and lytic enzymes, recruitment of inflammatory cells and osteoclast activation2 causing local inflammation and destruction of mineralized and nonmineralized periapical tissues,1 including collagen and other extracellular matrix components.3 Matrix metalloproteinases (MMPs) are zinc-dependent enzymes (endopeptidases), primarily responsible for the degradation of extracellular matrix macromolecules.4 Currently there are more than 23 known types of MMPs which are distributed into 6 groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and others.5 Several MMP family members, including MMP-1, -2, -3, -8, 9 and -13 are involved in the pathogenesis of periapical lesions.6–9 MMP-1 has been implicated as a key enzyme in the initiation of bone resorption,10 whereas MMP-2 and -9 are involved in the degradation of the extracellular matrix, mainly in the active stages of periapical lesion development.6 The production of MMPs are regulated by cytokines,4 tissue inhibitors of MMPs (TIMPs),11 as well as by matrix metalloprotreinase inducer (EMMPRIN). Also known as CD147 or basigin, EMMPRIN is a glycoprotein, a member of the immunoglobulin family, related to the highly glycosylated membrane.12 It is found in a wide range of tissues and expressed by normal, inflammatory and neoplastic cells.13–15 EMMPRIN can stimulate the production of MMPs-1, -2, -3, 7, -9, -13, and others13,16 and therefore participates in collagenolytic activity17 in physiological and pathological processes,13–15 including periodontal diseases.16,18–20 However, EMMPRIN participation in the periapical lesion development is still unknown. In order to better understand the molecular and cellular mechanisms that regulate the onset and progression of periapical lesion, the objective of this study is to evaluate the expression of EMMPRIN and its correlation with the expression of MMPs-1, -2 and -9 during the development of induced periapical lesion in mice.

2.

Materials and methods

All animal procedures are in accordance with the applicable ethical guidelines and regulation of the University’s Animal Research Ethics Committee, which approved the project (process number 08/2011). A total of 40 C57BL/6 wild-type mice were used; they were 7 to 8 week-old male mice, weighing on average 23 g.

2.1.

Periapical lesion induction

The protocol for the induction of periapical lesion was based on Silva et al.21 The mice were anaesthetized by intramuscular injection of 10% ketamine hydrochloride (150 mg/kg of body weight) and 2% xylazine hydrochloride (7.5 mg/kg of body weight) in the thigh and mounted on a jaw retraction board.

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The pulp chamber of the first lower molars of both sides were accessed with stainless-steel no. 1/4 round bur in a slowspeed handpiece and the mesial and distal root canals were explored with no. 08K file (Les Fils d’Auguste Maillefer S/A, Ballaigues, Switzerland) to remove the radicular pulp. The root canals were left exposed to the oral cavity for lesion induction. After 7, 14, 21 and 42 days, these mice were euthanized by anaesthetic overdose, as well as those from the control group (day 0), which had no crown opening. We used a total of 40 mice to obtain mandibles that were later separated in 80 hemimandibles (teeth), distributed as follows: 30 hemimandibles were immediately frozen at 70 8C for real-time RT-PCR (n = 6 hemimandibles for each time point—0, 7, 14, 21 and 42 days) and 50 hemimandibles were fixed in 10% phosphatebuffered formalin during 24 h for both mCT and histopathological analysis (n = 10 hemimandibles for each time point). The minimum sample size for each group (with 80% power and significance level of 5%) was identified as 6 for real-time RT-PCR and 10 for mCT and histopathological analysis for an expected difference of 30% between the groups.

2.2.

mCT

After fixation, 10 hemimandibles of each experimental period (0, 7, 14, 21 and 42 days) were prepared for mCT scanning (SkyScan 1176 in vivo, Skyscan, Kontich, Belgium). The scanning was performed at 80 kV, 300 mA, with an increment of 17.4 mm, 0.5 mm aluminium filter and 300-ms integration time. About 150 mCT images were acquired in the sagittal plane over the entire length of the mandible. The CTAn v.1.11.8 program (Skyscan) was used to select the mCT images that showed both the root canal and apical foramen of the distal root to measure the periapical lesion area in mm2. About 10 microtomographic mCT images per tooth were assessed, and the largest lesion area was used as the final value for each sample. All measurements were performed by one trained examiner who had no previous identification information about the groups assessed, and the measurements were carried out twice at different times.

2.3.

Histopathological analysis

After microtomographic scanning, the 10 hemimandibles were decalcified in an ethylenediaminetetraacetic acid (EDTA) solution at 4.13%, in pH 7.2 at 6 8C changed weekly for 30 days. The specimens were then submitted to routine histotechnical processing and embedded in paraffin. Longitudinal 5 mm thick serial sections were cut in a mesiodistal orientation along the periapical lesion size. The slices were stained with HE or submitted to immunohistochemistry to identify MMP-1, -2, -9 and EMMPRIN. All analysis were performed in the distal root of the mandibular first molar by one skilled examiner, blinded to the groups, using a Leica DMR microscope (Leica Microsystem Wetzlar Gmbh; Wetzlar, Germany) coupled to a Leica DFC 300FX camera (Leica Microsystems AG, Heerbrugg, Switzerland).

2.4.

Immunohistochemical analysis

The immunohistochemical (IHC) reactions were performed using the immunoperoxidase technique. Representative

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histological sections of each specimen were deparaffinized, hydrated, and immersed in 3% hydrogen peroxide for 20 min to inactivate endogenous peroxidase. Next, the sections were incubated overnight at 4 8C with the primary antibodies: antiMMP-1 (rabbit polyclonal antibody, Abbiotec, CA, USA, diluted 1:100); anti-MMP-2 (mouse monoclonal antibody, Santa Cruz Biotechnology Inc., CA, USA; diluted 1:100); anti-MMP-9 (goat polyclonal antibody; Santa Cruz Biotechnology Inc., CA, USA; diluted 1:100); anti-EMMPRIN (goat polyclonal antibody; Santa Cruz Biotechnology Inc., CA, EUA; diluted 1:100). Next, sections were incubated with secondary antibody followed by the streptavidin–biotin–peroxidase complex (ABC Kit 4000, Vecstain; Vector Laboratories Inc.) and 3,3’–diaminobenzidine solution (DAB; Sigma-Aldrich Corp., St Louis, MO, USA). Counterstaining was done with Harris’s haematoxylin. The negative control was treated in the same product, omitting the primary antibody. EMMPRIN and MMPs were evaluated qualitatively considering the presence/absence and localization of immunostaining in the radicular pulp and in the periapical region of the control teeth, and in the periapical region of the other groups. The areas stained by anti-EMMPRIN antibodies in the periapical region of all groups were also semi-quantitatively analyzed by two blinded skilled observers; any disagreements were discussed until consensus was reached. Three fields at 400 magnification around the root apex of the teeth were selected in three directions including the longitudinal axis of the root canal along the apical foramen and 458 to the longitudinal axis to the right and to the left.22 The EMMPRIN immunoexpression was analyzed by 0–4 scores based on the percentage of positively stained areas in each field: 0 = 0–10%; 1 = 10–24%; 2 = 25–49%; 3 = 50–74%; 4 =  75% (adapted from Fukuoka et al.23).

2.5.

Real-time RT-PCR

For the control group samples, tissue corresponding to the mesial and distal roots of the first lower molar (including radicular pulp) and surrounding structures (bone, periodontal ligament) were collected for real-time RT-PCR analysis. Using bone pliers, the tissues were excised in a block specimen. The gingiva, oral mucosa, and crown were removed and discarded. In groups with periapical lesions induction, the sample collected corresponded to the same tissue as that described above, including the periapical lesion and excluding the healthy radicular pulp, which was removed during the procedures necessary for periapical lesion induction. The entire amount of material collected was used for RNA extraction. The specimens were triturated with the liquid nitrogen and placed in a tube with 500 mL trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). The RNA extraction used the method that combines the use of trizol followed by partially using the extraction kit Illustra RNAspin Mini (GE Healthcare, Germany). One mg of RNA was used for complementary DNA confection in a standardized way for all groups using the Kit ImPron II Reverse Transcription System (Promega, Madison, WI, EUA) The b-actin, EMMPRIN, MMP1, MMP-2 and MMP-9 genes were detected and quantified by real-time RT-PCR using the SYBR Green (Go Taq1 qPCR— Master Mix, Promega). The primer sequences were designed

Table 1 – Sequences of the primers used. Gene

Sequence

b-Actin

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

EMMPRIN MMP-1 MMP-2 MMP-9

AACGAGCGGTTCCGATG GGATTCCATACCCAAGAAGGA CCTGCATCTTCCTTCCTGAG GACCAGTTTCGCAAGCTCTC GCCCTGATGTTTCCCATCTA ATGCTTAGGGTTGGGGTCTT ACCCAGATGTGGCCAACTAC AAAGCATCATCCACGGTTTC CAGCCGACTTTTGTGGTCTT GCTTCTCTCCCATCATCTGG

with the use of the Primer 3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and are listed in Table 1. Real-time RT-PCR samples were run in the Step One Plus Sequence Detector (Applied Biosystems, Foster City, CA, USA). Results were obtained as threshold cycle (Ct) values. Ct values were normalized with b-actin gene expression, following the equation 2 DDCt according to the manufacturer’s ‘‘User’s Bulletin no. 2 (PIN 4303859—Applied Biosystems)’’.

2.6.

Statistical analysis

The results of EMMPRIN and MMPs gene expression and periapical lesion area were analyzed by analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test. The results of EMMPRIN immunostaining were analyzed by Kruskal–Wallis test and Dunn post-hoc test. The Pearson correlation test was used to evaluate the correlation between the expression of EMMPRIN gene and MMPs genes in the active (7–14 days) and stable phase (21–42 days) of periapical lesion development. The correlation was moderate for r > 0.4 and strong for r > 0.7. The significance level of 5% was used. The data, when not specified, were expressed as mean and standard error of the mean.

3.

Results

3.1. lesion

Microtomographic morphometry of the periapical

The results of periapical lesion area are presented in absolute values of the periapical space area at the study-times, and in percentage-difference of this area in the time range of 0–7, 7– 14, 14–21 and 21–42 days (Fig. 1). The comparison of the absolute values of the periapical space area showed that there was no statistically significant bone loss at 7 days ( p > 0.05). At 14 days, the periapical lesion area was higher than at 7 days ( p < 0.05). At 21 and 42 days no statistically significant bone loss was observed in comparison to 14 days ( p > 0.05). The percentage-difference of the periapical lesion area was 34%, 83%, 1% and 18% in the periods of 0–7, 7–14, 14–21 and 21–42 days, respectively.

3.2.

Histopathological analysis

Seven days after procedures for periapical lesion induction, absence of the whole pulp area was observed, with the

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Fig. 1 – Two-dimensional microtomographic images representing the first lower molars in the control group—0 day (A) and 7 (B), 14 (C), 21 (D) and 42 (E) days after procedures for periapical lesion induction and contamination of the root canals. (F) Quantification of periapical periodontal space of the distal root. In the control group integrity of the lamina dura, periodontal ligament space and normal alveolar bone were observed. Alveolar bone loss and increased periapical lesion area were observed at 14, 21 and 42 days. The bar percentages represent the area difference of the periapical lesion, in percentage, in the periods of 0–7, 7–14, 14–21, 21–42 days. Groups marked with equal letters presented no statistically significant difference when compared using post-hoc test ( p > 0.05); groups marked with different letters presented statistically significant difference when compared using post-hoc test ( p < 0.05).

periodontal ligament space slightly increased and remarkable inflammatory cell infiltration; the cementum and alveolar bone appeared to be normal. At 14, 21 and 42 days the periapical ligament space was sharply increased and dense inflammatory infiltrate composed of mononuclear cells and polymorphonuclear cells distributed throughout the periapical lesion. The periapical lesion was characterized, in these periods, as unstructured periodontal ligament, alveolar bone resorption and cementum resorption areas (Fig. 2).

3.3.

Immunohistochemical analysis

The control group showed discrete and occasional EMMPRIM, MMP-1, -2 and -9 immunostaining in the periodontal ligament fibroblasts. MMP-2 was also intensely expressed in blood vessels of the periodontal ligament. EMMPRIN was strongly expressed in cells of the odontoblast layer of the control dental pulp and MMP-1 was moderately expressed in these cells (Fig. 3).

At 7, 14, 21 and 42 days after procedures for periapical lesion induction, intense immunostaining of EMMPRIN, MMPs-1, -2 and -9 was mainly observed in the region adjacent to the apical foramen and occasionally in other regions of the periapical lesion. There was intense immunostaining of EMMPRIN, MMP-1, -2 and -9 in polymorphonuclear and mononuclear cells and slight immunostaining in fibroblasts. The infiltrated cells were identified as macrophages, lymphocytes and neutrophils based on cell shape, size and shape of the nucleus (Fig. 3). The EMMPRIN immunoexpression in the periapical lesion was high at 7, 14, 21 and 42 days compared to its production in the periodontal ligament of the control teeth ( p < 0.05), as shown in Fig. 4.

3.4.

Expression of EMMPRIN, MMP-1, -2 and -9 genes

As shown in Fig. 5, EMMPRIN gene expression was higher in the control group than at 7, 14, 21 and 42 days ( p < 0.05). In

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Fig. 2 – Histological images representative of the distal root of the lower first molars of the control group—0 day (A) and 7 (B), 14 (C), 21 (D), and 42 (E) days after procedures for periapical lesion induction. The control group showed normal pulp coated with odontoblastic (Od) and predentin layer (Pd), periodontal ligament and alveolar bone integrity. At 7 days there was absence of the whole pulp, periodontal ligament lightly thickened with inflammatory cells, and bone and cement integrity. At 14, 21 and 42 days there was increased periodontal ligament space with dense inflammatory infiltrate distributed throughout the lesion region, and cementum (arrowheads) and alveolar bone (arrows) resorption. HE; Original magnification 100T and scale bars = 200 mm.

addition, at 42 days its expression was lower than at 7, 14 and 21 days ( p < 0.05). The MMP-1 gene expression was higher at 7, 14, 21 and 42 days than in the control group ( p < 0.05). The MMP-2 and -9 genes showed similar expression during the periapical lesion development; the expression of both genes were significantly higher at 14 days compared to control and periods of 7, 21 and 42 days of periapical lesion ( p < 0.05).

There was a significant moderate positive correlation between EMMPRIN and MMP-1 genes expression, besides significant strong positive correlation of EMMPRIN with MMP2 and -9 genes expression in the active phase (7 and 14 days) of periapical lesion development (r = 0.57, p = 0.031 for MMP-1; r = 0.76, p = 0.002 for MMP-2; r = 0.71, p = 0.004 for MMP-9). There was no correlation between EMMPRIN and MMPs genes

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Fig. 3 – Representative images of the control group and periapical lesions on day 14. In the control group there was a discrete and occasional staining of EMMPRIN (A and B), MMP-1 (E and F), MMP-2 (I and J) and MMP-9 (M and N) in the periodontal ligament fibroblasts (red arrows). MMP-2 was also intensely expressed in blood vessels (Bv) of the periodontal ligament. In the pulp of control teeth, odontoblasts (Od) strongly expressed EMMPRIN and moderately expressed MMP-1. After the procedures for periapical lesion induction, there was intense immunostaining of EMMPRIN (C and D), MMP-1 (G and H), MMP-2 (K and L) and MMP-9 (O and P) observed mainly in the region adjacent to the apical foramen and occasionally in other regions of the periapical lesion. EMMPRIN, MMPs-1, -2 and -9 showed strong expression in mononuclear (arrowheads) and polymorphonuclear (arrows) inflammatory cells, besides discrete expression in fibroblasts (red arrows). Original magnification 100T and scale bars = 200 mm in (A), (C), (E), (G), (I), (K), (M), (O), and the same figures in original magnification of 400T and bars = 50 mm in (B), (D), (F), (H), (J), (L), (N), (P).

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Fig. 4 – The EMMPRIN immunoexpression analysis in the control group—0 day and 7, 14, 21 and 42 days after procedures for periapical lesion induction. Bars represent the medians. Groups marked with equal letters presented no statistically significant difference when compared using post-hoc test ( p > 0.05); groups marked with different letters presented statistically significant difference when compared using post-hoc test ( p < 0.05).

expression in the stable phase (21 and 42 days) of the periapical lesion development ( p < 0.05).

4.

Discussion

This study showed for the first time the location, the temporal expression of EMMPRIN and its correlation with the expression of MMPs-1, -2 and -9, during the experimentally induced periapical lesion development in mice. The histopathological and microtomographic analysis confirmed the effectiveness of the experimental protocol used in the periapical lesion induction.21 Two distinct phases were detected: an active phase, from 7 to 14 days, when a significant difference of 83% was observed in the lesion area, followed by a chronic or stabilization phase at periods of 21 and 42 days, with no significant difference in lesion area.1,6,24 The EMMPRIN immunoexpression in the periapical region was higher in teeth with lesion than in the control ones. In the control teeth, the EMMPRIN was discrete and occasionally expressed in the periodontal ligament fibroblasts. At 7, 14, 21 and 42 days after the periapical lesion induction, intense immunostaining was observed in the inflammatory infiltrate adjacent to the apical foramen and in other inflamed regions of periapical lesion, besides a discrete staining in the fibroblasts. The EMMPRIN immunoexpression observed in the inflamed tissues of the periapical lesion is in agreement with studies that report that EMMPRIN is found at high levels

in the inflamed periodontal tissues18,19,25 and in the gingival crevicular fluid of teeth with periodontitis.26,27 Furthermore, periodontal therapies that reduce the inflammation of the periodontal tissues are associated with reduced EMMPRIN levels in the gingival crevicular fluid.27 The EMMPRIN expression by inflammatory cells and fibroblasts is consistent with reports that this molecule is produced by activated T cells,28 macrophages, monocytes,29 neutrophils30 and by inflammatory cells and fibroblasts in the gingival tissue of subjects with periodontitis.18,19 Using real-time RT-PCR, we found that the mRNA level of EMMPRIN was higher in the control group than in the teeth with periapical lesions at 7, 14, 21 and 42 days. The opposite results observed by comparing real-time RT-PCR and IHC semi-quantitative evaluations is probably because EMMPRIN expression may have been overestimated in the control tissues due to odontoblasts of the dental pulp producing this molecule.14 Furthermore, the EMMPRIN immunoexpression evaluation by IHC did not include pulp in the control group, but only the staining in the periapical region. It has been admitted that during the periapical lesion development the degradation of extracellular matrix components occurs in part due to the action of MMPs expressed by inflammatory cells, fibroblasts and endothelial cells.6,31 Indeed, the MMPs-1, -2 and -9 were immunoexpressed by inflammatory cells and fibroblasts at 7, 14, 21 and 42 days after the periapical lesions induction in the same regions with positive EMMPRIN staining. MMP-1 is considered a key enzyme in the initiation of bone resorption10 since it degrades the collagen of the unmineralized layer covering the bone surface, thereby allowing osteoclast bone-fixation.10,32 In addition, the collagen fragments generated lead to osteoclast activation at the beginning phase and in other bone resorption phases.10,32 It has been shown that MMP-1 expression increases in the active phase of periapical lesion development in rats33 and causes expansion of radicular cysts in humans.34 In the present study, the MMP-1 gene expression was higher than the control in the time periods studied, indicating their participation in the active and stable phases of the periapical lesion. It has been admitted that MMP-2 and -9 are involved in the degradation of the extracellular matrix, mainly in the active stages of periapical lesion development.6 Recent study in humans31 showed significantly higher activity of MMPs-2 and -9 in periapical lesions compared to healthy periodontal ligament, supporting their involvement in the pathogenesis of apical periodontitis. The expression of MMP-2 and -9 genes was high at 14 days along with a significant increase in lesion area, in contrast to a lower level compared to 14 days in the lesion stabilization periods (21 and 42 days). These results corroborate a previous study that reports the highest expression of these MMPs in the active development phase of periapical lesions and decreased expression during the lesion stabilization phase in rats.6 By stimulating the expression of MMPs and hence the degradation of the extracellular matrix, EMMPRIN is involved in the invasive property of malignant tumors13 and in other physiological and pathological processes.13–15,18 To the best of

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Fig. 5 – Expression analysis of the genes EMMPRIN (A), MMP-1 (B) -2 (C) and -9 (D) at 0, 7, 14, 21 and 42 days after the experimental induction procedure of the periapical lesion. Groups marked with equal letters presented no statistically significant difference when compared using post-hoc test ( p > 0.05); groups marked with different letters presented statistically significant difference when compared using post-hoc test ( p < 0.05).

our knowledge, this study showed for the first time that the expression of EMMPRIN gene positively correlates with the expression of MMPs-1, -2, and 9 in the active phase of periapical lesion development. These results suggest that EMMPRIN can be involved in regulating the expression of MMP-1, -2 and -9 and consequently in tissue destruction during the active phase of periapical lesion development. However, further studies are needed to elucidate the mechanisms involved in the EMMPRIN expression and its significance to the pathogenesis of periapical lesions. Finally, it could be concluded that there is a high expression of EMMPRIM mainly by the inflammatory infiltrate in the region adjacent to the apical foramen during periapical lesion development. Furthermore, the positive correlation with MMP-1, -2, and -9 during the first days after periapical lesion induction indicates that EMMPRIM may be involved in the active phase of periapical lesions development in mice.

Funding This study was supported by grants from Sa˜o Paulo Research Foundation (FAPESP) to G.F. (process no. 2008/58064-6 and no. 2011/19511-0) and by scholarship from CAPES to N.G.K.S.

Competing interests The authors deny any conflicts of interest in this study.

Ethical approval The study was approved by the Araraquara Dental SchoolUNESP Ethical Committee for Animal Research with process number 08/2011.

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Acknowledgements The authors are indebted to Dr. Marcos A. Rossi (in memorian) of Department of Pathology, Ribeira˜o Preto Medical School, University of Sa?o Paulo, for contribution in study design and contribution with reagents/materials/analysis tool and with analysis of data.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.archoralbio.2014.05.021.

references

1. Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med 2004;15(6):348– 81. 2. Stashenko P, Teles R, D’Souza R. Periapical inflammatory responses and their modulation. Crit Rev Oral Biol Med 1998;9(4):498–521. 3. Hannas AR, Pereira JC, Granjeiro JM, Tjaderhane L. The role of matrix metalloproteinases in the oral environment. Acta Odontol Scand 2007;65(1):1–13. 4. Verma RP, Hansch C. Matrix metalloproteinases (MMPs): chemical–biological functions and (Q)SARs. Bioorg Med Chem 2007;15(6):2223–68. 5. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003;92(8):827–39. 6. Corotti MV, Zambuzzi WF, Paiva KB, Menezes R, Pinto LC, Lara VS, et al. Immunolocalization of matrix metalloproteinases-2 and -9 during apical periodontitis development. Arch Oral Biol 2009;54(8):764–71. 7. Paula-Silva FW, da Silva LA, Kapila YL. Matrix metalloproteinase expression in teeth with apical periodontitis is differentially modulated by the modality of root canal treatment. J Endod 2010;36(2):231–7. 8. Matsui H, Yamasaki M, Nakata K, Amano K, Nakamura H. Expression of MMP-8 and MMP-13 in the development of periradicular lesions. Int Endod J 2011;44(8):739–45. 9. Menezes-Silva R, Khaliq S, Deeley K, Letra A, Vieira AR. Genetic susceptibility to periapical disease: conditional contribution of MMP2 and MMP3 genes to the development of periapical lesions and healing response. J Endod 2012;38(5):604–7. 10. Sasaki K, Takagi M, Konttinen YT, Sasaki A, Tamaki Y, Ogino T, et al. Upregulation of matrix metalloproteinase (MMP)-1 and its activator MMP-3 of human osteoblast by uniaxial cyclic stimulation. J Biomed Mater Res B: Appl Biomater 2007;80(2):491–8. 11. Ertugrul AS, Dursun R, Dundar N, Avunduk MC, Hakki SS. MMP-1, MMP-9, and TIMP-1 levels in oral lichen planus patients with gingivitis or periodontitis. Arch Oral Biol 2013;58(7):843–52. 12. Biswas C, Zhang Y, DeCastro R, Guo H, Nakamura T, Kataoka H, et al. The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer Res 1995;55(2):434–9.

13. Omi Y, Shibata N, Okamoto T, Obara T, Kobayashi M. The role of CD147 in the invasiveness of follicular thyroid carcinoma cells. Thyroid 2012;22(4):383–94. 14. Yang SY, Park BI, Kim HJ, Kang JH, Jung NR, Byun JD, et al. Differential expression of cyclophilin A and EMMPRIN in developing molars of rats. Anat Rec (Hoboken) 2012;295(1):150–9. 15. Dang Y, Li W, Tran V, Khalil RA. EMMPRIN-mediated induction of uterine and vascular matrix metalloproteinases during pregnancy and in response to estrogen and progesterone. Biochem Pharmacol 2013;86(6):734–47. 16. Yang D, Wang J, Ni J, Shang S, Liu L, Xiang J, et al. Temporal expression of metalloproteinase-8 and -13 and their relationships with extracellular matrix metalloproteinase inducer in the development of ligature-induced periodontitis in rats. J Periodontal Res 2013;48(4):411–9. 17. Kataoka H, DeCastro R, Zucker S, Biswas C. Tumor cellderived collagenase-stimulatory factor increases expression of interstitial collagenase, stromelysin, and 72-kDa gelatinase. Cancer Res 1993;53(13):3154–8. 18. Dong W, Xiang J, Li C, Cao Z, Huang Z. Increased expression of extracellular matrix metalloproteinase inducer is associated with matrix metalloproteinase-1 and -2 in gingival tissues from patients with periodontitis. J Periodontal Res 2009;44(1):125–32. 19. Liu L, Li C, Cai X, Xiang J, Cao Z, Dong W. The temporal expression and localization of extracellular matrix metalloproteinase inducer (EMMPRIN) during the development of periodontitis in an animal model. J Periodontal Res 2010;45(4):541–9. 20. Wang J, Yang D, Li C, Shang S, Xiang J. Expression of extracellular matrix metalloproteinase inducer glycosylation and caveolin-1 in healthy and inflamed human gingiva. J Periodontal Res 2013. doi: http://dx.doi.org/ 10.1111/jre.12095. 21. Silva MJ, Sousa LM, Lara VP, Cardoso FP, Junior GM, Totola AH, et al. The role of iNOS and PHOX in periapical bone resorption. J Dent Res 2011;90(4):495–500. 22. de Paula-Silva FW, Wu MK, Leonardo MR, da Silva LA, Wesselink PR. Accuracy of periapical radiography and conebeam computed tomography scans in diagnosing apical periodontitis using histopathological findings as a gold standard. J Endod 2009;35(7):1009–12. 23. Fukuoka M, Hamasaki M, Koga K, Hayashi H, Aoki M, Kawarabayashi T, et al. Expression patterns of emmprin and monocarboxylate transporter-1 in ovarian epithelial tumors. Virchows Arch 2012;461(4):457–66. 24. Aranha AM, Repeke CE, Garlet TP, Vieira AE, Campanelli AP, Trombone AP, et al. Evidence supporting a protective role for Th9 and Th22 cytokines in human and experimental periapical lesions. J Endod 2013;39(1):83–7. 25. Liu L, Li C, Cai C, Xiang J, Cao Z. Cyclophilin A (CypA) is associated with the inflammatory infiltration and alveolar bone destruction in an experimental periodontitis. Biochem Biophys Res Commun 2010;391(1):1000–6. 26. Emingil G, Tervahartiala T, Mantyla P, Maatta M, Sorsa T, Atilla G. Gingival crevicular fluid matrix metalloproteinase (MMP)-7, extracellular MMP inducer, and tissue inhibitor of MMP-1 levels in periodontal disease. J Periodontol 2006;77(12):2040–50. 27. Emingil G, Atilla G, Sorsa T, Tervahartiala T. The effect of adjunctive subantimicrobial dose doxycycline therapy on GCF EMMPRIN levels in chronic periodontitis. J Periodontol 2008;79(3):469–76. 28. Nabeshima K, Suzumiya J, Nagano M, Ohshima K, Toole BP, Tamura K, et al. Emmprin, a cell surface inducer of matrix metalloproteinases (MMPs), is expressed in T-cell lymphomas. J Pathol 2004;202(3):341–51.

archives of oral biology 59 (2014) 944–953

29. Major TC, Liang L, Lu X, Rosebury W, Bocan TM. Extracellular matrix metalloproteinase inducer (EMMPRIN) is induced upon monocyte differentiation and is expressed in human atheroma. Arterioscler Thromb Vasc Biol 2002;22(7):1200–7. 30. Wang CH, Dai JY, Wang L, Jia JF, Zheng ZH, Ding J, et al. Expression of CD147 (EMMPRIN) on neutrophils in rheumatoid arthritis enhances chemotaxis, matrix metalloproteinase production and invasiveness of synoviocytes. J Cell Mol Med 2011;15(4):850–60. 31. Dezerega A, Madrid S, Mundi V, Valenzuela MA, Garrido M, Paredes R, et al. Pro-oxidant status and matrix metalloproteinases in apical lesions and gingival crevicular fluid as potential biomarkers for asymptomatic apical

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periodontitis and endodontic treatment response. J Inflamm 2012;9(1):8. 32. Holliday LS, Welgus HG, Fliszar CJ, Veith GM, Jeffrey JJ, Gluck SL. Initiation of osteoclast bone resorption by interstitial collagenase. J Biol Chem 1997;272(35):22053–8. 33. Lin SK, Kok SH, Kuo MY, Wang TJ, Wang JT, Yeh FT, et al. Sequential expressions of MMP-1, TIMP-1, IL-6, and COX-2 genes in induced periapical lesions in rats. Eur J Oral Sci 2002;110(3):246–53. 34. Lin SK, Chiang CP, Hong CY, Lin CP, Lan WH, Hsieh CC, et al. Immunolocalization of interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in radicular cysts. J Oral Pathol Med 1997; 26(10):458–63.

Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases-1, -2 and -9 during periapical lesion development.

To evaluate the expression of matrix metalloproteinase inducer (EMMPRIN) and its correlation with the expression of matrix metalloproteinases (MMPs)-1...
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