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

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 concentrations of MMP-2, MMP-9, TIMP-1, and TIMP-2 in patients with Graves’ disease (GD), with and without GO, and their relationship with disease severity, as well as to evaluate how these concentrations change after successful treatment.


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. 2,3ECM metalloproteinases (MMPs) constitute a group of 28 zinc-dependent endopeptidases involved in the proliferation, migration, differentiation, angiogenesis, apoptosis, and host defense.Among MMPs, MMP-2 and MMP-9 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 interactions.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 -4.5][6][7] 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 MMP-9 but not MMP-2 serum concentration, whereas TIMP levels have not been examined. 8Therefore, the aim of this study was to assess the serum concentrations of MMP-2, MMP-9, TIMP-1, and TIMP-2 in patients with GD, with and without GO, and their relationship with disease severity, as well as to evaluate how these concentrations 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 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. 3Commercially 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 recommendations of the European Group on Graves' Orbitopathy (EUGOGO). 9One or more of the following clinical signs 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 20-60 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. 3This clinical and laboratory remission was achieved in all patients.No infections were observed at the time of parameter measurements.The control group consisted of 19 persons 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

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 concentrations of MMP-2 and MMP-9 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 MMP-9 concentration was significantly higher in patients with GO in comparison with GO-negative patients.Median serum concentrations 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, MMP-9, TIMP-1, and TIMP-2 concentrations significantly decreased after successful treatment.Moreover, the MMP-9/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 MMP-9 concentration and the serum level of TSHRAb antibodies, and a clinical activity score ≥4 according to EUGOGO (r = 0.265, p = 0.04; r = 0.8180, p = 0.0001, 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 considered to be constitutive (MMP-2) or inducible (MMP-9) in the quiescent tissue, many factors affect their synthesis.2][13] 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 concentrations 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 MMP-9 was significantly higher in GO patients compared with both GO-negative and normal individuals, and decreased after successful steroid treatment. 8The 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. 14oreover, Yoon et al. observed that quercetin, a flavonoid phytoestrogen, inhibited in vitro inflammation and concentrations of MMP-2 and MMP-9, 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 concentrations of all cytokines tested, as well as the MMP-9/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 persons without ocular manifestations 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 MMP-9 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   accumulation of the extracellular cell matrix in GO orbital fibroblasts stimulated with pro-inflammatory cytokines. 15,16The same authors demonstrated that quercetin inhibited MMP-2 and MMP-9 in orbital fibroblasts, which led to the prevention of chronic fibrosis in GO patients. 17However, it is not clear whether the concentration of MMP-2 or MMP-9 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 TSHRAb by this enzyme in the pathogenesis of the ocular manifestation of GD, and also suggests the possibility of a relationship 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 MMP-9 is more pronounced than the increase of its inhibitor TIMP1, and this finding strengthens the hypothesis of the role of MMP-9 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 sensitive 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 concentrations of MMP-9, MMP-2, TIMP-1, and TIMP-2 were elevated in patients with GO and decreased after the treatment, we found that only MMP-9 differentiates the patients with and without GO, and may be used as a marker of disease severity in patients with this manifestation of GD.

Table 1 .
Clinical and biochemical data of patients with GD