Gelatinases and Extracellular Matrix Metalloproteinase Inducer are associated with cyclosporine-A induced attenuation of periodontal degradation in rats

Gelatinases and Extracellular Matrix Metalloproteinase

Inducer (EMMPRIN) are Associated with Cyclosporine-A

Induced Attenuation of Periodontal Degradation in Rats

Martin Ming-Jen Fu,*, _{† Earl Fu,* Po-Jan Kuo,* Hsiao-Pei Tu,*}, ‡ _{Yu-Tang Chin,*}, §

Cheng-Yang Chiang,* Hsien-Chung Chiu*

* _{Department of Periodontology, School of Dentistry, National Defense Medical Center} and Tri-Service General Hospital, Taipei, Taiwan, ROC

† Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, MA

‡ Department of Dental Hygiene, China Medical University, Taichung, Taiwan, ROC § Institute for Cancer Biology and Drug Discovery, Taipei Medical University, Taipei,

Taiwan, ROC

Correspondence to: Dr. Hsien-Chung Chiu

Department of Periodontology School of Dentistry

National Defense Medical Center

PO Box 90048-507, Taipei, Taiwan, ROC Tel: +886-2-87927150; Fax: +886-2-87927145 Email: [email protected]

One Sentence Summary: Cyclosporine-A inhibits periodontal destruction via inhibition of gelatinases and EMMPRIN

Running Title: Cyclosporine-A inhibits gelatinases, EMMPRIN, and periodontitis

Figures: 6

Tables: 0

References: 47

ABSTRACT

Background: The present study aimed to exam the inhibitory effect of Cyclosporin-A

(CsA) on periodontal destruction and to further explore the correlations of CsA-induced

attenuation of periodontal bone loss with the expressions of gelatinases (i.e. matrix

metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)) and their

inducer, extracellular matrix metalloproteinase inducer (EMMPRIN).

Methods: Forty SD rats were randomly divided into four groups: control, CsA, ligature

(Lig), and ligature-plus-CsA (Lig+CsA) groups. CsA group received 10 mg/Kg/day of

CsA for 8 days. Lig group received silk ligature on selected molars. Lig+CsA group

received silk ligature and CsA treatment. The inhibitory effects of CsA on the

ligature-induced periodontal destruction was examined with micro-CT and histometric analysis to

analyze the amount of attachment loss, crestal bone loss, connective tissue attachment,

and the surface area with inflammatory cell infiltration. The effects of CsA on

ligature-induced expressions of gelatinases and EMMPRIN in gingival tissues were examined

with Western blotting and zymography, respectively.

Results: By micro-CT and histology, Lig+CsA group had significantly more periodontal

destruction than control and CsA groups, but less periodontal destruction than Lig group.

Consistent results were found for the expressions of gelatinases and EMMPRIN among

expression of gelatinases and EMMPRIN than Lig group.

Conclusion: CsA inhibited the expressions of gelatinase MMPs and EMMPRIN, and

partially prevented the periodontal destruction in ligature-induced experimental

periodontitis. The CsA-induced attenuation of periodontal bone loss was strongly

correlated positively with the expressions of MMP-2, MMP-9, and EMMPRIN in

gingiva.

KEY WORDS

Periodontitis; alveolar bone loss; gelatinases, matrix metalloproteinase 2; matrix

INTRODUCTION

Periodontitis is an immune-inflammatory disorder caused by microbial pathogens of

plaque that initiate host-mediated destruction of the periodontium. The microbial

pathogens and immune responses interact and initiate inflammatory cells to produce

pro-inflammatory mediators, such as cytokines, hydrolytic enzymes, reactive oxygen species,

and matrix metalloproteines (MMPs), and consequently leads to the destruction of the

surrounding connective tissue, periodontal attachment, and supporting alveolar bone.3-5

MMPs are a large family of proteases/peptidases characterized as key players in the

degradation of extracellular matrix and connective tissue proteins during periodontal

destruction.6 _{MMPs are classified into six groups, one of which is the gelatinase group.}

The gelatinases include MMP-2 (gelatinase A) and MMP-9 (gelatinase B), and have been

shown to be associated with periodontitis.7-15 _{Increased expressions of MMP-2, MMP-9,}

and their potent inducer, extracellular matrix metalloproteinase inducer (EMMPRIN, also

known as CD147, basigin, BSG, and neurothelin) have been found in the gingival

crevicular fluid7-9 _{and gingiva}10-13 _{from patients with chronic periodontitis. The}

over-expression of these proteins has also been found to be decreased significantly after

periodontal therapies.

EMMPRIN is a widely expressed plasma membrane-bound glycoprotein that

is an upstream inducer of several MMPs17 _{and stimulates the production of MMP-1, -2,}

-3, and -9.18-20 _{In chronic periodontitis, EMMPRIN has been shown to mediate the}

collagenolytic balance to favor the production and activation of MMPs.

Cyclosporin-A (CsA), a fungus-derived cyclic peptide, is a widely used

immunosuppressant which helps prevent organ transplant rejection due to its suppressive

action on specific T-cell subpopulations. One of the common side effects of the

administration of CsA is gingival overgrowth. The synthetic and catabolic effects of CsA

on gingival tissues have also been broadly observed. CsA is able to affect fibroblast

proliferation, promote abnormal accumulation of extracellular matrix components in the

gingival lamina propria, and influence the catabolic enzymes of the extracellular

matrix.25-29 _{The suppression mechanism of the inhibition of CsA on the activity of MMPs}

in gingiva has also been investigated recently. During gingival overgrowth, CsA has been

shown in vitro to inhibit the expression of MMP-230-34 _{and MMP-9}30 _{by our laboratory}

and others.

It is a common belief that the inflamed and overgrown gingiva induced by CsA

could become a bacterial plaque reservoir, therefore aggravating the progression of

periodontitis. However, advanced periodontal destruction in the area with CsA-induced

gingival overgrowth is not frequently observed for unknown reasons.

the initial periodontal breakdown by bringing an imbalance in the alveolar bone

homeostasis.35 _{The present study, which uses the model of ligature-induced experimental}

periodontitis in rats, aimed to examine the inhibitory effect of CsA on periodontal

destruction and to further explore the correlations of CsA-induced attenuation of

periodontal bone loss with the expressions of gelatinases (i.e. MMP-2 and MMP-9), and

MATERIALS AND METHODS

Animal and Experimental Design

The animal research protocol was approved by the Institutional Animal Care and

Use Committee of National Defense Medical Center. In order to examine the effect of

CsA on the experimental periodontal destruction after the placement of silk ligature, forty

6-week-old male SD rats were randomly divided into four groups (i.e. control, CsA,

ligature, and ligature-plus-CsA groups) with ten rats in each group. The rats in the control

group were fed daily with mineral oil which is the solvent of CsA, whereas the rats in

CsA group received CsA∥ (10 mg/Kg in mineral oil) once a day for 8 consecutive days.

The rats in the ligature (Lig) group were fed daily with mineral oil and had 3-O silk

suture¶ placed on the cervical necks of maxillary second and mandibular first molars. The

rats in ligature-plus-CsA (Lig+CsA) group received silk ligature as those in ligature

group but also received the same CsA regimen as the CsA group did. At the end of the

experiment, which was 8 days after, all of the animals were sacrificed with carbon

dioxide inhalation. In each rat, the gingival tissues at the palatal side of maxillary second

molars and around mandibular first molars were taken and pooled for Western blot (to

examine the expression of EMMPRIN) and zymography (to examine the expressions of

MMP-2 and MMP-9). The remaining maxillary specimens were fixed in 4%

histology.

Micro-CT Imaging

All of the maxillary block biopsies were subjected to micro-CT imaging by a

multi-modality preclinical imaging system.# The x-ray tube was operated at an accelerated

potential of 75 kVp with a beam current of 120 μA. The field of view (FOV) for

micro-CT was fixed to 61.44 mm leading to 2X magnification of image. The micro-micro-CT

images were taken under fly mode with 1024 projections and 1 frame per projection

to achieve a voxel size of 120x120x120 μm3_{. The micro-CT data were}

reconstructed** prior to being visualized and analyzed by software.†† The micro-CT

bone level, which is defined as the distance from cemento-enamel junction (CEJ) to the

coronal level of alveolar bone crest (ABC), was measured at twelve sites. These sites

included the mesio-buccal, mid-buccal, disto-buccal, mesio-palatal, mid-palatal, and

disto-palatal sites of the right and left maxillary second molars.

Gelatin Zymography

The release of gelatinases (including pro-MMP-2, MMP-2, and pro-MMP-9) from

gingival tissues was evaluated using gelatin zymography. After grinding, the proteins in

the gingival homogenates were measured using protein assay.‡‡ The proteins of the

SDS-polyacrylamide gel containing 0.1% gelatin. Equal amounts of protein were loaded into

each lane of the gel. After electrophoresis, the gel was placed into renaturing buffer

(2.5% Triton-X100), shaken gently to remove SDS, and then incubated in developing

buffer for 16 hours. The gel was stained with 2.5% Coomassie Brilliant Blue. The latent

and active forms of MMP-2 were detected as 72 kDa and 62 kDa bands, respectively,

while the latent form of MMP-9 was detected as 92 kDa band. The gel images were then

scanned and detected quantitatively.§§

Western Blotting

Protein samples were resolved on a 10% Sodium Dodecyl Sulfate (SDS) polyacrylamide

gel. Twenty micrograms of protein were loaded in each well with 4 sample buffer, and

the protein samples were resolved by electrophoresis at 100 V for 2 hours. The resolved

proteins were transferred∥∥from the polyacrylamide gel to membrane.¶¶ The membranes

were blocked with a solution with 5% nonfat milk and 1% Tween 20 in Tris-buffered

saline, and incubated with primary antibodies to EMMPRIN## and α-tubulin##

overnight. The proteins were detected using HRP-conjugated secondary antibodies and

reagent.*** The images were then visualized and recorded using autoradiography.

Histopathology and Histometry

On the mesial surfaces of second molars in each of rats, the distance between the

following histometric landmarks were performed (Fig. 2A): CEJ to ABC (indicating

alveolar bone loss), CEJ to the coronal level of epithelial cells (JEc)(indicating

attachment loss), and the apical level of epithelial cells (JEa) to ABC (indicating CT

attachment). The amount of surface area with inflammatory cell-infiltrated connective

tissue (ICT) in a zone of 0.14 mm2 _{of sub-epithelial gingiva on the mesial surfaces of}

maxillary second molars of each rat was also measured.

Statistical Analysis

Repeated-measures analysis of variance (ANOVA) with Duncan’s test for post-hoc

analysis was used to evaluate the influence of the inter-subject factor (i.e. CsA or ligature

treatment), as well as the intra-subject factors from all four groups together (including the

mesial, middle or distal site, the buccal or palatal surface, and the left or right side) on the

micro-CT bone level (the distance from CEJ to ABC). Regression analysis with

correlation coefficient was used to determine the associations of alveolar crest bone loss

(the distance from CEJ to ABC) to the expressions of MMPs and EMMPRIN in all of the

animals with four different treatments. Results are expressed as means and standard

deviations. P < 0.05 was considered as significant.

¶ Surgical silk sutures, UNIK, Taiwan

# FLEX Triumph for platformX-O, Gamma Media-Ideas, Inc., Northridge, CA ** Triumph XO, Gamma Media-Ideas, Inc., Northridge, CA

†† VIVID, Gamma Media-Ideas, Inc., Northridge, CA ‡‡ Bicinchoninic acid protein assay, Pierce, Rockford, IL

§§ Transilluminator/SPOT, Diagnostic Instruments, Sterling Heights, MI ∥∥ Hoefer semi-dry transfer system, Hoefer, Inc., Holliston, MA

¶¶ Millipore Immobilon-PSQ transfer PVDF membranes, Millipore, Billerica, MA ## Abcam, Cambridge, MA

RESULTS

Cyclosporine-A Impedes Periodontal Destruction

By micro-CT, more alveolar bone loss was observed at the ligature sites of the Lig

and the Lig+CsA groups, compared to those at the same sites of the control or CsA group

(Fig. 1A). However, the bony destruction was less severe in the Lig+CsA group than in

the Lig group. By the repeated measures analysis of variance, the mean micro-CT bone

levels (CEJ-ABC) were significantly different among the four animal groups and among

the examining sites, as well as between the buccal and palatal/lingual locations (Fig. 1B).

Statistically similar bone crests levels were recorded in the control and CsA groups after

the post-hoc analysis, whereas the distance from CEJ to ABC in Lig+CsA group was

statistically longer than the control and CsA groups but shorter than the Lig group.

By histology, gingival inflammation and alveolar bone loss were easily observed in

the ligature group, but were not as severe in the control and CsA groups (Fig. 2A). Less

severe periodontal inflammation and destruction was also noted in the Lig+CsA group

when compared with the Lig group. Histometric data demonstrated a consistent findings

showing that significantly less alveolar bone loss, attachment loss, and inflammation

(ICT area) were noted in the Lig+CsA group when compared with the Lig group (Figs.

2B, 2C, 2D). However, significantly less connective tissue attachment (JEa-ABC) was

Cyclosporine-A-induced Attenuation of Periodontal Destruction was Correlated with Expressions of Gelatinases, and Their Inducer, EMMPRIN.

By zymography, the gelatinolytic activities of pro-MMP-9, pro-MMP-2 and MMP-2

among four animal groups were examined (Figs. 3A-3D). The activities of the examined

MMPs were all significantly increased in the Lig and Lig+CsA groups compared with the

control and CsA groups. The MMPs activities in Lig+CsA group were significantly lower

those in Lig group, but higher than those in the control and CsA groups. Correlation

analysis also demonstrated very strong positive correlations of the activities of

pro-MMP-9, pro-MMP-2 and MMP-2 with alveolar bone loss (CEJ-ABC) in both micro-CT (Figs.

4A-4C) and histometrical analysis (Figs. 4D-4F).

By Western blotting, the protein expression of EMMPRIN was significantly

stronger in gingiva obtained from the Lig and Lig+CsA groups when compared with

those from the control and CsA groups; whereas the expression of EMMPRIN in the

Lig+CsA group was significantly weaker when compared to that in the Lig groups (Fig.

5). The correlation analysis also showed a strong positive correlation of the activities of

EMMPRIN with the amount of alveolar bone loss (CEJ-ABC) in both micro-CT (Fig.

DISCUSSION

The local tissue breakdown of periodontitis is caused by a series of

plaque-associated immuno-inflammatory processes in the periodontium. The inflamed and

overgrown gingiva induced by CsA could become a bacterial plaque reservoir and

aggravate the progression of periodontitis. However, the severity of periodontal

breakdown is often inconsistent with the severity of enlargement, inflammation, and

plaque accumulation observed in the patients with CsA-induced gingival overgrowth. As

seen in a previous radiographic observation,35 _{this present study found a correspondingly}

inhibitory effect of CsA on the periodontal destruction using micro-CT and histometric

analysis. The present study, which uses a ligature-induced periodontitis rat model, was

the first in vivo study demonstrating that CsA-induced attenuation of periodontal bone

loss is correlated positively with the expressions of MMP-2, MMP-9, and their inducer,

EMMPRIN. It is also the first study illustrating an inhibitory effect of CsA on

EMMPRIN in gingiva.

MMPs are known to play a major role in the degradation of extracellular matrix,

basal membrane, and connective tissue proteins. MMPs has been shown to be associated

with embryonic development, wound healing, and some pathophysiological responses

such as arthritis, fibrosis, and inflammation.38 _{MMP-2 and MMP-9 belong to the}

synthesized by fibroblasts, keratinocytes, endothelial cells, macrophages, platelets,

osteoblasts, and chondroblasts, whereas MMP-9 is usually produced by neutrophils,

keratinocytes and macrophages.3 _{Increased expressions of MMP-2}11-13 _{and MMP-9 have}

been found in patients with chronic periodontitis which, is consistent with the findings of

the present study (Fig. 3 negative and positive controls and Fig. 4).

EMMPRIN is a transmembrane glycoprotein originally identified on the surfaces of

tumor cells. Its expression on tumor cells may induce tumor progression and invasion by

triggering the production or release of MMPs. EMMPRIN is expressed on all leukocytes,

keratinocytes, endothelial cells, and platelets,42 _{and is now best known as an inducer of}

MMPs (including MMP-2 and MMP-9).20 _{The ability of EMMPRIN to stimulate MMP}

production suggests the possibility that this molecule is associated with several

pathophysiological tissue modulatory processes and tissue remodeling, including

asthma-mediated lung inflammation, rheumatoid arthritis, sclerosis, myocardial infarction, and

ischemic stroke.42-44 _{In periodontitis, increased expression of EMMPRIN has also been}

shown in patients with chronic periodontitis, and in a ligature-induced periodontitis

model. The results of the present study showing a significantly increased expression of

EMMPRIN (Fig. 5 negative and positive controls and Fig. 6) were also consistent with

the findings in the previous studies.

of EMMPRIN in their gingival tissues has been examined once.47 _{However in that study,}

both of the CsA and healthy control patients showed a very strong intensity of peroxidase

staining for EMMPRIN. Our laboratory has also found that the total protein expression of

EMMPRIN in gingival fibroblasts which received CsA was unchanged when compared

to that without CsA in vitro; however, its membranous expression was decreased after

CsA stimulation (unpublished data). In the current study, we demonstrated that the

expression of EMMPRIN did decrease when CsA was given in the ligature-induced

periodontitis (Figs. 5 and 6).

Recent in vitro studies from our laboratory and others have also shown that CsA can

inhibit the productions of MMP-230-34 _{and MMP-9}30 _{in gingival fibroblasts. The in vivo}

results in the present study (Figs. 3 and 4) explain the attenuated effect of CsA on

periodontal bone loss, and are consistent with the inhibitions of MMP-2 and MMP-9 seen

in the previous in vitro studies. In addition, a less severe inflammatory cell infiltration

was observed in the CSA+Lig group compared to the Lig group (Fig. 2C). It might imply

that in the patients with CsA-induced gingival overgrowth and periodontitis, the

interaction between the gingival fibroblasts and inflammatory cells involves not only the

synergic activities from CsA but also the metabolic activities caused by the CsA. It has

been suggested that CsA may inhibit the initial periodontal breakdown, because in the

imbalance in the alveolar bone homeostasis in rats.35

In order to elucidate the possible role of gelatinase MMPs and EMMPRIN on

CsA-induced attenuation of periodontal destruction, the expressions of MMP-2, MMP-9, and

EMMPRIN in gingiva, as well as their correlations with the micro-CT and histologic

tissue losses were examined (Figs. 3 to 6). The results suggest that the CsA-induced

attenuation of periodontal destruction might be involved with the regulation of

MMP/CD147 pathway. However, the roles of EMMPRIN and its activities on MMPs in

CsA-induced gingival overgrowth are still unclear and require further detailed

investigations.

CONCLUSIONS

The current study demonstrated inhibitory effects of CsA on gelatinases (i.e.

MMP-2 and MMP-9), EMMPRIN, and periodontal destruction using a ligature-induced

periodontitis rat model. The study results also showed a strong positive correlation of

CsA-induced attenuation of periodontal bone loss with the expressions of 2,

MMP-9, and EMMPRIN in gingiva.

ACKNOWLEDGMENTS

National Defence (DOD-C101-12-4) and the National Science Council

(NSC-100-2314-B-039-053), Republic of China. The authors report no conflicts of interest related to this

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Figure 1. Effect of CsA on the ligature-induced alveolar bone loss. (A) presents the

reconstructed 3D images from the computerized tomography for maxillae viewed from the buccal or palatal direction in the control, CsA, ligature, and CsA-plus-ligature groups. Asterisks indicate the molar with the ligature. (B) shows the influence of the between-subject factors (the treatment groups) and the within-subject factors (the mesial/middle/distal site, the buccal/palatal surface, and the right/left jaw) on the micro-CT bone level. (*, †, and ‡: significantly different subsects obtained by the post hoc Duncan test) (Lig + CsA: ligature-plus-CsA group).

1A

Figure 2. Effect of CsA on ligature-induced periodontal tissue destruction. (A)

histographs present the inter-proximal periodontal tissue between the first and second maxillary molars from the control, CsA, ligature, and ligature-plus-CsA groups (H & E stain). Arrows indicates the most coronal level of alveolar bone crest (ABC). The micrographs in the bottom row (40X) represent higher magnifications of images in the top row (20X). (B to E) present the comparisons of results by histometry, including periodontal tissue loss (CEJ-JEc distance), histological bone level (CEJ-ABC distance), severity of gingival tissue inflammation (ICT), and length of connective tissue attachment (JEa-ABC distance) among the four animal groups. (*, †, and ‡: significantly different subsects obtained by the post-hoc Duncan test) (mm2₎

2A

2B

2C

Figure 3. Effect of CsA on ligature-induced gelatinolytic activities of pro-MMP-2, MMP-2, and pro-MMP-9 in gingiva. (A) shows the patterns of gelatinolytic activities at

92 (pro-MMP-9), 66 (MMP-2), and 72 (pro-MMP-2) kDa. (B to D) show the comparisons of their optical intensities of the MMPs among the treatment groups. Data are expressed as means and standard deviations. The experiment was repeated four times. (*, †, and ‡: significantly different subsects obtained by the post-hoc Duncan test)(Lig: ligature group; Lig + CsA: CsA-plus-ligature group )

3A

3B

Figure 4. Correlation of the gingival expressions of gelatinase MMPs with severity of alveolar bone loss in ligature-induced periodontitis. (A to F) correlation analysis

revealed a strong, positive correlation of the activity of MMPs (in pixels) with the micro-CT distance of CEJ-ABC (A to C), and the histological distance of CEJ-ABC (D to F).

4A

4B

4C

Figure 5. Effect of CsA on ligature-induced protein expressions of EMMPRIN in gingiva. (A and B) present the protein expressions of EMMPRIN (CD-147) in gingivae

of rats obtained from four treatment groups examined by Western blotting. Data are expressed as means and standard deviations. (*, †, and ‡: significantly different subsects obtained by the post-hoc Duncan test)

5A

5B

Figure 6. Correlation of the gingival expressions of EMMPRIN with severity of alveolar bone loss in ligature-induced periodontitis. (A and B) correlation analysis

revealed a strong positive correlation of the activity of EMMPRIN (in pixels) with the micro-CT distance of CEJ-ABC (Fig. 6A), and the histological distance of CEJ-ABC (Fig. 6B).