Elevated serum concentrations of metalloproteinases (MMP-2, MMP-9) and their inhibitors (TIMP-1, TIMP-2) in patients with Graves orbitopathy

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1 Original papers Elevated serum concentratio of metalloproteinases (MMP-2, ) and their inhibitors (TIMP-1, TIMP-2) in patients with Graves orbitopathy Katarzyna Kapelko-Słowik 1, A F, Mirosław Słowik 2, A C, Marek Szaliński 2, A, B, Jarosław Dybko 1, B, C, Dariusz Wołowiec 1, E, Iwona Prajs 1, C, Anna Bohdanowicz-Pawlak 3, B, Monika Biernat 4, C, D 1, 5, A F, Donata Urbaniak-Kujda 1 Department of Hematology, Blood Neoplasms and Bone Marrow Traplantation, Wroclaw Medical University, Poland 2 Department of Ophthalmology, Wroclaw Medical University, Poland 3 Department of Endocrinology, Diabetology and Isotope Treatment, Wroclaw Medical University, Poland 4 Department of Microbiology, Wroclaw Medical University, Poland 5 Department of Cosmetology, Wroclaw Physiotherapy College, Poland A research concept and design; B collection and/or assembly of data; C data analysis and interpretation; D writing the article; E critical revision of the article; F final approval of the article Advances in Clinical and Experimental Medicine, ISSN (print), ISSN (online) Adv Clin Exp Med. 2018;27(1): Address for correspondence Katarzyna Kapelko-Słowik kks9999@wp.pl Funding sources None declared Conflict of interest None declared Received on September 14, 2016 Reviewed on December 21, 2016 Accepted on February 14, 2017 Abstract Background. Graves orbitopathy (GO), also known as thyroid-associated ophthalmopathy, is characterized by dramatic tissue reactivity. Both inflammation and tissue remodeling characterize the clinical course of GO. Some data has been found regarding the association of MMPs and TIMPs in GO. Material and methods. Serum concentratio of, MMP-2, TIMP-1, and TIMP-2 were determined by ELISA method. Objectives. Forty-eight patients (34 females, 14 males, with median age 51.5 years) with GD and hyperthyroidism were enrolled in the study. In 28 patients, active, moderate-to-severe grade orbitopathy was diagnosed. The aim of this study was to assess the serum concentratio of MMP-2,, TIMP-1, and TIMP-2 in patients with Graves disease (GD), with and without GO, and their relatiohip with disease severity, as well as to evaluate how these concentratio change after successful treatment. Results. Median serum concentratio of MMP-2 and were significantly higher in all patients with GD as well as in the subgroup with GO than in the control group. Median serum concentratio of TIMP-1 and TIMP-2 were significantly higher in all patients with GD than in controls. The same significant differences were observed in the subgroups with and without GO in comparison with controls. The GO subgroup showed a significant positive correlation between the concentration and the serum level of TSHRAb antibodies, and a clinical activity score 4 according to EUGOGO. Conclusio. In our study we found that only differentiates the patients with and without GO, and may be used as a marker of the disease severity in patients with this manifestation of GD. Key words: Graves disease, matrix metalloproteinases, tissue inhibitors of metalloproteinases, orbithopathy DOI /acem/68991 Copyright 2018 by Wroclaw Medical University This is an article distributed under the terms of the Creative Commo Attribution Non-Commercial Licee (

2 100 K. Kapelko-Słowik, et al., MMP-2, TIMP-1 and TIMP-2 and GO Introduction Graves orbitopathy (GO), also known as thyroid-associated ophthalmopathy, is characterized by dramatic tissue reactivity. It is clinically proven in 10 45% of patients with Graves diseases (GD). 1 The clinical course of GO is characterized by both inflammation and tissue remodeling. Inflammation dominates at the beginning of GO, followed by the remodeling of the orbital connective tissue, including the accumulation of extracellular matrix (ECM) macromolecules and fibrosis. 2,3 ECM metalloproteinases (MMPs) cotitute a group of 28 zinc-dependent endopeptidases involved in the proliferation, migration, differentiation, angiogenesis, apoptosis, and host defee. Among MMPs, MMP-2 and are very important for collagen degradation. The concentration of MMPs is very low in the quiescent tissue, but their expression can be increased by inflammatory cytokines, hormones, growth factors, and cell interactio. A number of factors regulate these processes taking place in the extracellular matrix, including natural tissue inhibitors of metalloproteinase (TIMPs). Four TIMPs are known: TIMP-1,-2,-3, and -4. TIMP-1 and TIMP-3 can inhibit proteolytic activity of, andtimp-2,-3, and -4 bind and inactivate MMP Some data has been found regarding the association of MMPs and TIMPs in GO. It was found that glucocorticosteroid administration in patients with GO significantly decreased but not MMP-2 serum concentration, whereas TIMP levels have not been examined. 8 Therefore, the aim of this study was to assess the serum concentratio of MMP-2,, TIMP-1, and TIMP-2 in patients with GD, with and without GO, and their relatiohip with disease severity, as well as to evaluate how these concentratio change after successful treatment. Patients Forty-eight patients (34 females, 14 males, with median age 51.5 years) diagnosed with GD and hyperthyroidism between October 2010 and October 2012 at the Department Table 1. Clinical and biochemical data of patients with GD Parameter GD (all patients) GO GO-negative Number of patients Age [years], median Gender [f/m] 34/14 20/8 14/6 FT3 [pmol/l], median (25Q-75Q) 8.0 ( ) 8.2 ( ) 7.3 ( ) FT4 [pmol/l], median (25Q-75Q) 18.6 ( ) 19.9 ( ) 17.4 ( ) TSH [U/L], median (25Q-75Q) 0.1 ( ) 0.2 ( ) 0.06 ( ) TSHRAb [U/L], median (25Q-75Q) ( ) ( ) ( ) f females; m males; FT3 free triiodothyronine; FT4 free thyroxine; TSH thyroidstimulating hormone (thyrotropin); TSHRAb thyroid-stimulating hormone receptor antibodies, GD Graves disease; GO Graves orbitopathy. of Endocrinology, Diabetes and Isotope Treatment of Wroclaw Medical University (Poland) were enrolled in the study. GD diagnosis was determined using a clinical examination, laboratory results and thyroid gland ultrasonography. 3 Commercially available agents were used to determine thyroid-stimulating hormone (TSH), thyroxine (FT4), and triiodothyronine (FT3) levels. Serum concentration of thyroid-stimulating hormone receptor antibodies (TSHRAb) was analyzed by ELISA. Table 1 presents the patients characteristics. All of the patients were established ophthalmologically evaluated. In 28 patients, active, moderate-to-severe grade orbitopathy was diagnosed. Severity and activity of GO were established based on the recommendatio of the European Group on Graves Orbitopathy (EUGOGO). 9 One or more of the following clinical sig were observed: lid retraction 2 mm, moderate-to-severe orbital soft tissue involvement, proptosis 3 mm, permanent or periodic diplopia. According to the EUGOGO, a clinical activity score of 4/7 indicates active GO. 10 Patients in the current study presented with conjunctival injection, edema of the eyelids, and chemosis. Additionally, patients with clinically active orbitopathy were tested with magnetic resonance imaging (MRI) and ultrasonographic examination of the orbital muscles. Normal thyroid function was achieved using a thyreostatic drug (thiamazole Thyrozol) beginning with mg per day and decreasing the dose based on clinical and biochemical parameters. Supportive medication was propranolol (20 40 mg per day). The duration of treatment varied from 12 to 18 months. After reaching normal thyroid function, patients with active GO were given methylprednisolone intravenously, 3 times per week over a period of 4 weeks. This regimen was followed by oral prednisone starting with 60 mg and gradually decreasing the dose during 6 weeks. In all patients, marker analysis was repeated after successful treatment. In the GO subgroup, full remission of ocular symptoms was achieved, and was confirmed clinically and by ultrasonography. In patients without GO, the measure of successful treatment was the normalization of hormonal parameters and the remission of the clinical symptoms of hyperthyroidism. 3 This clinical and laboratory remission was achieved in all patients. No infectio were observed at the time of parameter measurements. The control group coisted of 19 perso without endocrine, neoplastic or inflammatory pathological changes, and matching the age and gender of the patient population. Methods Serum from whole peripheral blood was collected from patients at diagnosis and after successful treatment, during remission. All sera were kept in 70 C until

3 Adv Clin Exp Med. 2018;27(1): assessment. Serum concentratio of, MMP-2, TIMP-1, and TIMP-2 were determined by ELISA (R&D Systems Inc., Minneapolis, USA), according to the manufacturer s protocol. All samples and standards were measured in duplicate. Minimum detectable concentratio were 0.16 ng/ml (MMP-2), ng/ml (), 0.08 ng/ml (TIMP-1), and ng/ml (TIMP-2). Serum TSHRAb concentration was measured at the time of diagnosis by ELISA (Bio Vendor, Brno, Czech Republic) according to the manufacturer s protocol. Values <0.4 U/L were coidered negative. Statistical analysis The results were analyzed by the Mann-Whitney U test for uncorrelated data, the paired sample test, and the Spearman s rank correlation test. Results Median serum concentratio of MMP-2 and were significantly higher in all patients with GD as well as in the subgroup with GO before treatment than in the control group. This observation was not found in GO-negative patients regarding MMP-2. Moreover, median concentration was significantly higher in patients with GO in comparison with GO-negative patients. Median serum concentratio of TIMP-1 and TIMP-2 were significantly higher in all patients with GD before treatment than in controls. The same differences were observed in the subgroups with GO and without GO in comparison with controls. The data is presented in Table 2. Additionally, in the GO and GO-negative subgroups, median MMP-2,, TIMP-1, and TIMP-2 concentratio significantly decreased after successful treatment. Moreover, the /TIMP-1 ratio was significantly higher in all patients and in the groups with GO and without GO than in controls, but we did not observe ratio normalization after treatment. The data is presented in Table 3. It is worth noting that the subgroup with GO showed a positive correlation between the concentration and the serum level of TSHRAb antibodies, and a clinical activity score 4 according to EUGOGO (r = 0.265, p = 0.04; r = , p =, respectively). The data is presented in Fig. 1 and 2. Discussion MMPs play a key role in ECM remodeling and are involved in a variety of processes including inflammation, migration, differentiation, angiogenesis, and fibrosis. Although the expression of some MMPs is coidered to be cotitutive (MMP-2) or inducible () in the quiescent tissue, many factors affect their synthesis. Therefore, the clinical significance of MMPs, in particular MMP-2 and, has been revealed in many pathological conditio such as neoplasms, autoimmune diseases, and chronic inflammation. 4 6 Increased production of MMP-2 and appears to be a useful marker of several autoimmune disorders and neoplasms (e.g., multiple myeloma) The involvement of MMPs in pathological processes includes matrix degradation and an imbalance between MMPs and their specific inhibitors, TIMPs. TIMPs play important roles in maintaining the delicate balance between ECM production and disposal. Changes in their concentratio are associated with tissue dysfunction. The participation of these metalloproteinases as well as their inhibitors, TIMP1 and TIMP9, in the pathogenesis of ocular manifestation of GD, i.e., Graves orbitopathy (GO), has not been clearly established. Myśliwiec et al. reported that serum concentration of was significantly higher in GO patients compared with both GO-negative and normal individuals, and decreased after successful steroid treatment. 8 The MMP-2 concentration remained unchanged. In another report, the authors described an increase of the production of TIMP-1 in GO orbital fibroblasts after stimulation by interleukin 1β. 14 Moreover, Yoon et al. observed that quercetin, a flavonoid phytoestrogen, inhibited in vitro inflammation and Table 2. Comparison of median [M (25Q-75Q)] concentratio of MMP-2,, TIMP-1, TIMP-2, and the /TIMP-1 ratio in the serum of GD, GO and GO-negative patients before treatment and controls No. Patients MMP-2 TIMP-1 TIMP-2 /TIMP-1 1 GD (n = 48) (95 296) ( ) ( ) ( ) 3.85 (0.7 16) 2 GO (n = 28) (95 296) ( ) ( ) ( ) 4.80 ( ) 3 GO-negative (n = 20) ( ) ( ) ( ) ( ) 3.7 ( ) 4 controls (n = 19) ( ) ( ) ( ) 92.1 ( ) 2.1 ( ) p 1 vs 4 2 vs 4 3 vs 4 2 vs n number of patients; GD Graves disease; GO Graves orbitopathy; not significant; MMP-2 metalloproteinase-2; metalloproteinase-9; TIMP-1 tissue inhibitor of metalloproteinase-1; TIMP-2 tissue inhibitor of metalloproteinase

4 102 K. Kapelko-Słowik, et al., MMP-2, TIMP-1 and TIMP-2 and GO accumulation of the extracellular cell matrix in GO orbital fibroblasts stimulated with pro-inflammatory cytokines. 15,16 The same authors demotrated that quercetin inhibited MMP-2 and in orbital fibroblasts, which led to the prevention of chronic fibrosis in GO patients. 17 However, it is not clear whether the concentration of MMP-2 or allows for the differentiation between patients with and without an ocular manifestation. In particular, is it not clear which pathogenic mechanisms are involved in the development of ocular symptoms observed in some patients with GD. Therefore, the aim of the present study was to evaluate the serum Table 3. Comparison of median [M (25Q-75Q)] concentratio of MMP-2,, TIMP-1, TIMP-2, and the /TIMP-1 ratio in the serum of GD, GO and GO-negative patients, before and after treatment, and controls / TIMP-1 TIMP-2 TIMP-1 MMP-2 Number Patients 1 GD before treatment (n = 48) (95 296) ( ) ( ) ( ) 3.85 (0.7 16) 2 GD after treatment (n = 20) ( ) ( ) ( ) 92.4 ( ) 2.9 ( ) 3 GO-negative before treatment (n = 20) ( ) ( ) ( ) ( ) 3.7 ( ) 4 GO-negative after treatment (n = 10) ( ) ( ) ( ) 91.2 ( ) 2.8 ( ) 5 GO before treatment (n = 28) (95 296) ( ) ( ) ( ) 4.80 ( ) 6 GO after treatment (n = 10) ( ) ( ) ( ) 92.3 ( ) 2.9 ( ) 7 controls (n = 19) ( ) ( ) ( ) 92.1 ( ) 2.1 ( ) vs 2 3 vs 4 5 vs 6 2 vs 7 4 vs 6 p n number of patients; GD Graves disease; GO Graves orbitopathy; not significant; MMP-2 metalloproteinase-2; metalloproteinase-9; TIMP-1 tissue inhibitor of metalloproteinase-1; TIMP-2 tissue inhibitor of metalloproteinase-2. concentratio of MMP-2 and, and their inhibitors, TIMP-1 and TIMP-2, in patients with GD, with and without GO, both in the active phase and after successful treatment. We determined that serum concentratio of all cytokines tested, as well as the /TIMP-1 ratio, were significantly higher in the whole group of GD patients, and also in the sub-groups with and without GO, than in control subjects. However, only the MMP9 serum concentration was significantly more elevated in the patients with GO than in perso without ocular manifestatio of GD. It may be thus hypothesized, in line with the above quoted reports of Myśliwiec et al. and Yoon et al., that this enzyme is involved in a specific way in the pathogenesis of GO. The positive correlation we found between the concentration and TSHRAb and the disease activity according to the EUGOGO in the GO subgroup provides an additional argument in favor of a particular role played EUGOGO Fig. 1. Positive correlation between the concentration and clinical activity score 4 according to EUGOGO in patients with GO (r = , p = ) metalloproteinase-9. 4 Fig. 2. Positive correlation between the concentration and the serum level of TSHRAb antibodies in patients with GO (r = 0.265, p = 0.04) metalloproteinase-9; TSHRAb thyroid-stimulating hormone receptor antibodies. 5 TSHRAb 6

5 Adv Clin Exp Med. 2018;27(1): by this enzyme in the pathogenesis of the ocular manifestation of GD, and also suggests the possibility of a relatiohip between its release to the blood and the activity of the disease. The significant increase of the MMP9/ TIMP1 ratio we found in all subgroups of patients as compared to healthy controls deserves particular attention. It may indicate that, although the concomitant rise of both metalloproteinases and their inhibitors occurs in GD with and without GO, the increase in is more pronounced than the increase of its inhibitor TIMP1, and this finding strengthe the hypothesis of the role of in the development of GD with or without orbitopathy. Our findings showing that the serum concentration of all cytokines tested significantly decreased after successful treatment of all subgroups of patients suggest that these substances not only are involved in the pathogenesis of GD but also their production correlates with clinical activity of the disease. It is of interest that TIMP1 and the MMP9/TIMP1 ratio are the only parameters which remain significantly elevated in GD patients after successful treatment as compared to healthy subjects. Therefore, their usefulness as the most seitive markers of the disease should be further evaluated. Conclusion To the best of our knowledge, our study is the first analysis of the combined determination of serum concentration of 2 metalloproteinases and their inhibitors in GD with and without orbitopathy, in active disease and after successful treatment. Although the serum concentratio of, MMP-2, TIMP-1, and TIMP-2 were elevated in patients with GO and decreased after the treatment, we found that only differentiates the patients with and without GO, and may be used as a marker of disease severity in patients with this manifestation of GD. 7. Lambert E, Dassé E, Haye B, Petitfrère E. TIMPs as multifacial protei. 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