The ADAM12 is upregulated in synovitis and postinflammatory fibrosis of the synovial membrane in patients with early radiographic osteoarthritis

Joint Bone Spine 81 (2014) 51–56

Available online at www.sciencedirect.com

Original article

The ADAM12 is upregulated in synovitis and postinflammatory fibrosis of the synovial membrane in patients with early radiographic osteoarthritis Irina Kerna a,∗ , Kalle Kisand b , Siim Suutre c , Maret Murde d , Ann Tamm e , Jaanika Kumm f , Agu Tamm a a

Department of Internal Medicine, University of Tartu, L. Puusepa 6-222, 51014 Tartu, Estonia Department of Immunology, University of Tartu, Tartu, Estonia Department of Anatomy, Histology and Embryology, University of Tartu, Tartu, Estonia d Department of Pathology, Tartu University Hospital, Tartu, Estonia e Department of Sports Medicine and Rehabilitation, University of Tartu, Tartu, Estonia f Department of Radiology, University of Tartu, Tartu, Estonia b c

a r t i c l e

i n f o

Article history: Accepted 7 March 2013 Available online 9 April 2013 Keywords: Knee osteoarthritis Synovitis ADAM12 Gene expression

a b s t r a c t Objective: To investigate the possible association between ADAM12 (disintegrin and metalloproteinase domain12) expression in the synovium and the histological synovitis of patients with radiographic knee osteoarthritis (rKOA). Methods: The synovial biopsy samples were harvested from 44 subjects with chronic knee complaints during arthroscopy. In all subjects, the radiographs of both knee joints were performed for rKOA assessment. Histological features of synovitis were graded 0–3 in synovial samples. Messenger RNA (mRNA) of two ADAM12 splice variants [ADAM12-S(hort) and ADAM12-L(ong)] and the identical region for both–ADAM12-B(oth) were measured by real-time reverse transcription-PCR in all synovial samples (TaqMan® gene expression assay). Immunohistochemical staining of the synovial membrane with ADAM12 antibody was performed in 42 subjects. Results: ADAM12 mRNA was expressed in all synovial samples, whereas the main part of overall expression consisted of its long isoform (ADAM12-L). ADAM12 protein expression was detected in 80% of the synovial samples and correlated with mRNA expression (␳ = 0.30, P < 0.05). The expression of ADAM12 mRNA and protein in synovium correlated with the severity of histological synovitis (␳ = 0.28, P < 0.05 for ADAM12-B mRNA, R2 = 0.20, P < 0.05 for ADAM12 protein). Out of several features of synovitis the expression level of both splice variants correlated only with the grade of fibrosis in the synovium (␳ = 0.30, P < 0.05 for ADAM12-L and ␳ = 0.33, P < 0.05 for ADAM12-S). Conclusions: ADAM12 is upregulated in the synovial tissue during synovitis on mRNA and protein level. We suggest that ADAM12 could be implicated in the development of KOA-associated synovitis, especially in the occurrence of postinflammatory fibrosis. © 2013 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.

1. Introduction A variety of causes – ageing, genetics, metabolic and mechanical factors – are all major contributors to osteoarthritis (OA) onset and progression [1]. An increasing body of knowledge shows that OA is a complex disorder that involves the whole joint, encompassing, in addition, the cartilage and the synovial membrane, as well as the underlying bone, ligaments and muscle [2]. It is increasingly appreciated that inflammation of the synovial membrane is common in early and late OA [3,4]. The main histopathological features of the OA synovium include hypertrophy and hyperplasia with an increase in the number of synovial lining cells and

∗ Corresponding author. Tel.: +372 56 24 30 45; fax: +372 742 21 68. E-mail address: [email protected] (I. Kerna).

infiltration of the sublining tissue with scattered foci of mononuclear cells [5,6]. These cells are an abundant source of proinflammatory mediators, like cytokines, matrix metalloproteases and as well as growth factors [1,7]. Therefore, inflammation in OA is interpreted as a major factor associated with the risk of both progression of cartilage degradation and signs and symptoms of diseases [8]. Moreover, chronic synovitis can lead to epithelial mesenchymal transition and further to fibrosis of the synovial membrane, resulting in altered function of synovial membrane [1,9,10]. To this day, there is no generally accepted molecular description of the pathogenetic pathway of synovial inflammation. Multiple questions such as involvement of definite genes and nature of molecular interactions within the synovial pannus still remain unclear. Analysis of the synovial fluid of patients with early OA reveals an abundance of proteins and demonstrates a substantial

1297-319X/$ – see front matter © 2013 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2013.03.007

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I. Kerna et al. / Joint Bone Spine 81 (2014) 51–56

difference in protein composition compared to healthy subjects [11]. The matrix metalloproteinases, A Disintegrin And Metalloproteinase (ADAMs) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTSs) are the main proteolytic enzymes that regulate extracellular matrix turnover in the cartilage [12]. According to published data, a member of the ADAM gene family – ADAM12 – could be implicated in the pathogenesis of OA [13,14]. Polymorphisms of ADAM12 gene were reported to be associated with knee OA (KOA) development and progression [15,16]. Nevertheless, these results are not consistent as some other studies failed to replicate this association in other populations or races [17,18]. However, no meta-analysis or functional studies aiming to elucidate the susceptibility of ADAM12 to OA have been performed. The ADAM12 is a catalytically active metalloproteinase, which is expressed mainly in remodelling and fast-growing tissues [19]. The ADAM12 has been shown to be involved in growth factor shedding [20], cell adhesion and fusion [21,22], which suggest also its role in inflammatory and immune reactions. Therefore, ADAM12 has been shown to be upregulated in different cancer types [23], pregnancy disorders [24] as well in human OA cartilage [13,15]. There is limited data reflecting the expression pattern and behaviour of ADAM12 in OA elsewhere than the cartilage. The single available study has reported a detectable expression of ADAM12 in a minority of OA synovial samples [25]. However, the relation of ADAM12 to synovial membrane inflammation, as well as its possible contribution to KOA aggravation has not been studied. The aim of the present study was to investigate the possible relation of ADAM12 to histological synovitis. We defined the expression level of ADAM12 mRNA and protein in synovial biopsy samples and evaluated the associations of ADAM12 expression with features of histological synovitis and traits of radiographic knee OA (rKOA). 2. Methods 2.1. Tissue samples and patients The study group consists of 44 subjects (20 men), aged 32–60, mean 46.7 years, who underwent knee arthroscopy in Tartu University Clinics during years 2007–2010 due to chronic knee problems (several months until years). Patients with inflammatory arthropathies were not included in the research. Arthroscopic diagnosis was divided as injury of the medial or lateral menisci (n = 30), injury of ligaments (anterior cruciate, medial and lateral collateral ligaments, n = 8), and chondropathy (n = 6). Before the arthroscopy, all subjects participated in an in-depth clinical and radiological examination. Radiographs were obtained for the tibioand patellofemoral joint of both knees and were evaluated separately according to the grading system of Nagaosa et al. [26]. Details of the assessment are described previously [16]. The synovial tissue samples (weight 10–50 mg) were harvested from the synovial capsule (from macroscopic lesion) using biopsy forceps (Piranha Rongeur, Atlantech) for the histological evaluation and mRNA extraction. In 10 out of 44 patients the second sample of the tissue was obtained from a macroscopically unchanged synovium (suprapatellar recessus). The study was approved by the Ethical Committee of Human Research, Tartu University, and informed consent was obtained from all patients before arthroscopy. 2.2. RNA extraction 2.2.1. RNA extraction from the synovial membrane Obtained biopsies were stabilized in 300 ␮L of RNAlaterTM (Ambion, Inc., Austin, TX) solution overnight at 4 ◦ C and stored at −80 ◦ C until RNA extraction. All synovial tissue samples were grinded after thawing using T10 basic ULTRATURRAX® dispenser

(IKA® Werke GmbH & Co. KG, Staufen, Germany) and additionally homogenized with the syringe (needle G20). Total RNA was isolated from the homogenized synovial tissue samples using Qiagen Rneasy® Mini kit or Qiagen Rneasy® Fibrous Tissue Mini kit (QIAGEN, Valencia, CA), according to the provided manufacturer’s manuals. 2.2.2. RNA quality assessment The yield of total RNA was detected by NanoDrop® NT-1000 spectrophotometer (NanoDrop Technologies, Inc., Montchanin, DE, USA) and RNA integrity was analyzed by Agilent RNA 6000 Nano Chip on Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity index of total RNA from synovial samples was in the range 6.1–10.0. 2.3. Synthesis of cDNA The reverse transcription was performed using oligo-dT18 primers and Superscript III reverse transcriptase (Invitrogen Ltd, Paisley, UK), according to the manufacturer’s manual. cDNA was stored at −80 ◦ C until used in quantitative PCR. 3. TaqMan real-time PCR TaqMan quantitative PCR was conducted to measure the expression of ADAM12. Three different TaqMan® gene expression assays (Hs00185774 m1, Hs00222216 m1 and Hs01106104; Applied Biosystems, Foster City, CA) were selected for the detection of the expression of ADAM12-isoform I (ADAM12-Long or ADAM12-L), isoform II (ADAM-Short or ADAM-S), and total ADAM12 (ADAM12B, assay for identical regions of both isoforms). Human ␤-actin endogenous control (Primer-Limited) (Applied Biosystems) was used as a housekeeping gene. The quantitative real-time PCR assays were performed by ABI PRISM 7900 HT SDS (Applied Biosystems) in 384-well plate format according to the standard protocol [27]. The PCR reaction was performed in three parallels. The data were analyzed using the Sequence Detection Systems software, version 2.2.2 (Applied Biosystems). The real-time-PCR (RT-PCR) reactions were performed as multiplex (the housekeeper and gene of interest in the same well). The relative quantification levels for the gene expression were calculated using the 2-CT method (CT = crossing points, cycle number where the fluorescence crossed the threshold 0.1): CT = CT (target gene) − CT (reference gene); CT = CT (patients) − CT (reference) [28]. An acceptable standard deviation between the parallels was assigned as 0.2 and, in case of mean CT over 36 cycles, as 0.4. The cDNA of a non-pathological synovial specimen was used as a reference sample for all other synovial biopsies. 3.1. Evaluation of synovial membrane 3.1.1. Histological assessment The paraffin blocks for microscopical investigation were available for 42 patients. The biopsy samples obtained from two subjects were recognized unsuitable for histological evaluation and were therefore excluded from examination. Histological evaluation was performed by a clinical pathologist on formalin-fixed paraffinembedded sections stained by haematoxylin-eosin and Van Gieson. The microscopic features of synovitis were evaluated according to the system offered by Loeuille et al. [29]. Six parameters were evaluated separately:

I. Kerna et al. / Joint Bone Spine 81 (2014) 51–56

Radiographic KOA grade 0 1 2 3

5 (2) 29 (14) 9 (4) 1 (0)

BMI: body mass index; SD: standard deviation.

features and in two patients with advanced rKOA, histological synovitis was absent. It is remarkable that all moderate and severe cases of synovitis were found in the early (grade 1) rKOA group. Thus, the histological synovitis in our group was present in the majority of patients (86%), whereas all severe cases belonged to the early rKOA group. 4.2. The expression level of A Disintegrin And Metalloproteinase12 mRNA in synovial tissue We found that all three isoforms of ADAM12 are expressed in synovial tissue. The gene expression detection rate in synovial tissue was 100% for ADAM12-B, 96% for ADAM12-L, and 84% for ADAM12-S. The expression pattern of different isoforms of ADAM12 mRNA in synovial tissue is presented on Fig. 1. In the majority of the investigated samples, the expression of ADAM12-B exceeded the expression of both isoforms. However, in 13 subjects, the relative expression of ADAM12-L was higher than ADAM12-B. The proportion of secretable ADAM12-S was found to be significantly lower, 5 4 3 2 1

A D A

M A

M

12

12

-L

-S

0

D

The histological synovitis was found in 36 subjects out of 42 (Table 1). The degrees of the synovitis were divided as follows: mild synovitis in 29 patients, moderate in six, and severe (synovitis grade 3) in one patient. No features of histological synovitis were described in six investigated subjects. The rKOA was diagnosed in 39 of the 44 participants: early OA changes were found in 29 subjects and the late stages of the disease in 10 subjects (Table 1). There were no significant differences between the ages or BMI [P = non significant (NS)] of the subjects with and without rKOA features. There was no correlation between grades of rKOA and severity of histological synovitis (P = NS). The synovial inflammation was present in three subjects out of five (60%) without and in 33 (84%) patients with rKOA features. In three subjects with early KOA

6 (2) 29 (14) 6 (2) 1 (1)

A

4.1. Microscopical synovitis and radiographic knee osteoarthritis in the study group

Histological synovitis grade 0 1 2 3

-B

4. Results

28.5 ± 5.0 29.3 ± 4.5 27.9 ± 5.2

12

Spearman rank correlation test was used to determine the association between mRNA relative expression level (2-CT), grade of histological synovitis, and protein expression in the synovial membrane. The linear regression model was used for an analysis of the association between IHC staining and global grade of histological synovitis. As the data did not fit normal distribution, Wilcox exact test (WET) was used to compare gene expression in patients’ groups with different grades of rKOA. All analyses were carried out in the R-environment ver. 2.4.0 (The R Foundation for Statistical Computing, Boston, MA).

BMI (mean ± SD) All Males Females

M

3.2. Statistical analysis

46.7 ± 6.0 47.2 ± 5.4 46.3 ± 6.5

A

3.1.2. Immunohistochemistry (IHC) Deparaffinised sections were treated with 3% H2 O2 to inactivate endogenous peroxidase and then with Dako REAL Antibody Diluent (S2022; Dako Denmark A/S, Glostrup, Denmark) to block non-specific binding. After blocking, sections were incubated with mouse monoclonal antibody to ADAM12 (ab56366; Abcam Ltd.) overnight at 4 ◦ C. The primary antibody concentration was 3 ␮g/mL. Visualization of the primary antibodies was performed using commercial kit “Dako REALTM EnVisionTM Detection System, Peroxidase/DAB+, Rabbit/Mouse” (K5007; Dako Denmark A/S). The washing steps in-between were done in phosphate-buffered saline (PBS), which contained 0.07% of Tween 20 as a detergent. Toluidine blue (Applichem, Darmstadt, Germany) was used for background staining. Samples were evaluated in a semiquantitative manner by two examiners as follows: no staining = 0, mild staining = 1 (a few stained cells in field; magnification 40×), moderate staining = 2 (more than five stained cells), and strong staining = 3 (more than 10 stained cells). No immunohistochemical staining was noted in negative control samples where the primary antibody was omitted.

Age (mean ± SD) All Males Females

D

Each parameter was scored in ranges of 0–3. The sum of scores based on the six described parameters gives the global grade of histological synovitis, which was classified as absent (grade 0), mild (grade 1), moderate (grade 2), or severe (grade 3).

Table 1 Main characteristics of study group, presence of diagnosed histological synovitis and radiographic knee osteoarthritis (rKOA). For histological synovitis and rKOA data is presented as number of investigated subjects, in brackets is given a number of male subjects.

A

number of synovial lining cells; subsynovial infiltration by lymphocytes and plasma cells; surface fibrin deposition; congestion related to blood vessel vasodilatation and/or proliferation; • fibrosis; • perivascular oedema.

mRNA expression

• • • •

53

synovial tissue Fig. 1. The mRNA expression level of isoform I (long splice variant-A Disintegrin And Metalloproteinase12-L), isoform II (short secreted splice variant-A Disintegrin And Metalloproteinase-S) and variant identical for both isoform (A Disintegrin And Metalloproteinase12-B) in investigated synovial samples. The data present intraindividual variation of A Disintegrin And Metalloproteinase12-isoforms and ADAM12-B is analyzed as reference.

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Fig. 3. The expression of A Disintegrin And Metalloproteinase12 protein in investigated synovial samples. A. The ADAM12 was mainly expressed in synovial macrophages (black arrow) and fibroblasts (dashed arrow, magnification 40×). B. The synovial expression of A Disintegrin And Metalloproteinase12 mRNA and protein (based on immunohistochemistry) correlated significantly (rho-Spearman’s rank correlation).

Fig. 2. The features of histological synovitis and correlation of A Disintegrin And Metalloproteinase12 mRNA expression with synovial membrane inflammation. (A) The synovial infiltration by mononuclear cells (magnification 40×, black arrow). (B) Fibrosis (asterisk). (C) The overall expression of A Disintegrin And Metalloproteinase12 mRNA correlated with grade of histologic synoviatis in synovium. The severity of fibrosis of synovial membrane correlated with mRNA expression of both splice isoforms-A Disintegrin And Metalloproteinase12-L (D) and A Disintegrin And Metalloproteinase12-S (E) (rho-Spearman’s rank correlation).

representing only 4% of the overall expression of ADAM12 in the synovium. Taken together, ADAM12 mRNA was expressed in all investigated samples, whereas from two splice variants in the synovial tissue the long form (ADAM12-L) was predominantly present. 4.3. Association of A Disintegrin And Metalloproteinase12 mRNA expression with histological synovitis and radiographic knee osteoarthritis We found a weak but significant correlation between the expression of ADAM12-B and the grade of histological synovitis (␳ = 0.28, P < 0.05; Fig. 2). From two genders this association was significant only in males (␳ = 0.49, P < 0.05). A separate analysis of different phenotypic parameters of histological synovitis reveals that the expression level of both splice variants-ADAM12-L (␳ = 0.30, P < 0.05) and ADAM12-S (␳ = 0.33, P < 0.05) weakly but significantly correlated with the grade of fibrosis of the synovial membrane (Fig. 2). The relative expression values of ADAM12 in macroscopically unchanged and inflamed synovial tissue samples were similar. Additionally, there was no difference in measured ADAM12 mRNA expression between subjects with and without radiographic features of rKOA. According to the above results in our study group, the expression of ADAM12 mRNA group was upregulated in histological synovitis and correlated with the grade of fibrosis. 4.4. A Disintegrin And Metalloproteinase12 protein expression in the synovial membrane IHC staining reveals ADAM12 protein expression predominantly in macrophage and fibroblast cells in 80% of investigated synovial samples (Fig. 3). The ADAM12 protein expression (based on IHC) was in correlation with ADAM12-B mRNA expression (␳ = 0.30, P < 0.05; Fig. 3). From two splice variants of ADAM12 mRNA correlation with IHC staining was noticed predominantly for the short isoform (␳ = 0.38, P < 0.05). Observed correlations between ADAM12 protein and mRNA expressions were stronger in male

subjects compared to the whole group (␳ = 0.70, P < 0.001 for ADAM12-B and ␳ = 0.77, P < 0.01 for ADAM12-S). Additionally, ADAM12 protein expression was related to global grade of histological synovitis (R2 = 0.11, P < 0.05). The above results support the possible involvement of ADAM12 in synovial inflammation processes, which was observed earlier on the mRNA level. 5. Discussion This is the first study to investigate the possible association between ADAM12 and OA-associated synovitis. Our results demonstrate clearly that ADAM12 is expressed in the synovial membrane and that the expression of this gene is upregulated during synovial inflammation. Notably, this association was found not only at gene level (as mRNA expression) but also regarding the presence of a large amount of ADAM12 protein in the inflamed synovium. In addition, our results underline the importance of synovial inflammation in the course of OA, which is consistent with an increasing body of knowledge showing that inflammatory synovitis could be an early feature of OA and is not restricted to patients with the end-stage disease [30–32]. Moreover, in some forms of OA the molecular and biochemical changes in the affected synovial joint tissue advanced the pre-radiographic phase up to 2–3 years, indicating that synovial inflammation could even pre-date cartilage changes in several cases [6,33]. Our present data are consistent with this hypothesis. We also found prominent histological synovitis cases more frequently in patients with early rKOA than in the case of the advanced disease. This could also explain why grade of rKOA (based mainly on cartilage changes) did not correlate with severity of histological synovitis or expression of mRNA of ADAM12 in our study. The implication of ADAM12 in synovial membrane inflammation has not yet been investigated and only one study has reported the relatively low expression of ADAM12 (13% of biopsies) in OA samples [25]. In contrast, we found much higher expression of both ADAM12 mRNA (100%) and protein (80%) in the investigated synovial membrane biopsies. This discrepancy could be related to differences in the primers used for detection of expression rate: only the ADAM12-L isoform was measured in Komiya et al. study [25] while both splice variants were measured in our research. Naturally, there exist two splice variants of human ADAM12, giving rise to two different protein isoforms: ADAM12-L (a transmembrane form) and ADAM12-S (a secreted form). The longer splice variant, ADAM12-L, contains pro-a metalloprotease, disintegrin, cysteine-rich, EGF-like and transmembrane domains, whereas the shorter secreted form lacks the transmembrane and cytoplasmic domains [34]. Both ADAM12-isoforms are multifunctional zinc-dependent proteases with the ability to shed

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membrane-anchored proteins like cytokines, growth factors and their receptors. It has been proposed that cleavage of these substrates may regulate availability of bioactive molecules and thereby also regulate inflammation, tissue vascularization or remodelling [34]. Indeed, according to published data, ADAM12 appears to modulate mesenchymal cell differentiation and could therefore be involved in remodelling of different tissues [19]. Animal studies have shown that the long-term overexpression of ADAM12 is associated with inflammation but also with accelerated fibrosis and adipogenesis [35]. However, development of the synovial fibrosis depends on several regulators. In an experimental model its persistence is enhanced by simultaneous administration of TGFbeta and connective tissue growth factor [36]. Up-regulation of ADAM12 mRNA on fibrotic samples of the synovium observed in our material also suggests possible involvement of ADAM12 in fibrosis-associated synovial membrane remodelling. This finding is in line with a recent patent description, which suggests the use of ADAM12 as a new regulator for inflammation-induced fibrosis [37]. The authors of this patent proposed that blocking of ADAM12 by special inhibitors may open up new possibilities to prevent or even to treat inflammation-induced fibrosis-related complications like liver cirrhosis, scleroderma, heart and pulmonary fibrosis, atherosclerosis and asthma [37,38]. In the light of our and other results, it seems reasonable to set the hypothesis that inhibitors of ADAM12 could serve as drug candidates for modulating the course of OA. Our study has some limitations. First of all, because of ethical reasons, we did not have control samples of synovial tissues from subjects without any history of knee joint problems. In some studies, control samples for histology were obtained during autopsy but for evaluating mRNA expression, fresh samples from organ donors are needed. Additionally, the quality of several biopsy samples was insufficient for reliable histological evaluation. Indeed, joint replacement surgery would definitely provide more material with better quality, in the early phase of the disease, arthroscopic biopsy, providing a small amount of tissue, is the single possible modality with its limitations. Moreover, the design of this study did not allow us to specify the molecular mechanisms behind the possible implication of ADAM12 in the development processes of synovial fibrosis. Further studies are needed for the elaboration of ADAM12 gene’s role in development of synovitis and postinflammatory fibrosis. In summary, the results of our study enhance the understanding that synovitis is an integral component of KOA and that the expression of ADAM12 is associated with synovitis already in early phases of the disease. Moreover, we propose that ADAM12 could be associated with development of the synovial fibrosis and thereby impair the normal function of synovial lining. A better understanding of the biological and molecular mechanisms of synovial inflammation and of the role of ADAM12 in it could lead to the development of new therapeutic approaches, thereby potentially decelerating cartilage degradation. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We gratefully acknowledge orthopaedists Dr. T. Saluse, Dr. A. Pintsaar, Dr. T. Tein, Dr. A. Kukner of Tartu University Clinics for harvesting synovial tissue samples, pathologist Dr. M. Reintam of Tartu University Clinics for performing histological evaluation, Dr. U. Kööbi for coordination of survey, assistant A. Krips for helping in

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collection of biopsies specimens and Mrs. H. Karo and M. Vija for their assistance in creation of study database. Funding sources: this study was supported by a target financed project No. SF0180012s11 from the Estonian Ministry of Education and Research.

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