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Inoyatov Dilshod Anvarovich, researcher of Republican research and practical medical center of dermatovenerology and cosmetology, Republic of Uzbekistan Muydinov Otabek Khusanovich, assistant of Tashkent medical academy, Republic of Uzbekistan Yuldosheva Navruza Gaybullayevna, magistr of Tashkent medical academy, Republic of Uzbekistan Kalandarov Aslbek Ergashogli, magistr of Tashkent medical academy, Republic of Uzbekistan E-mail: [email protected]
THE ACTIVITY OF MATRIX METALLOPROTEINASES AND THEIR INHIBITORS IN PATIENTS WITH SCLERODERMA
Abstract: A study of the level of matrix metalloproteinases (MMP-2, MMP-7, MMP-9) in the serum of 29 patients with systemic and 35 patients with limited scleroderma showed a decrease in their activity against the increase in their inhibitors (TIMP-1 and TIMP-2), especially in patients with systemic scleroderma. The level of serum TGF-^1 increased significantly, and was also more pronounced in patients with systemic scleroderma. Correlation analysis of the relationship between MMPs and TIMP parameters with TGF-^ showed strong feedback with MMP-2, MMP-7 and MMP-9, a direct strong association with TIMP-1 and TIMP-2, more pronounced in the systemic course of the disease.
Keywords: systemic and limited scleroderma, matrix proteinases, matrix metalloproteinase inhibitors, transforming growth factor of fibroblasts.
The scleroderma (SD) takes the second place in the structure of modern dermatology. The scleroderma pathogenesis is not fully determined. Among the main causes of sclerotic changes in a skin, activation of fibro-genesis processes due to the extracellular matrix disturbance is being discussed [9; 10]. Structural components of extracellular matrix proteins are collagen, elastin, pro-teoglicans and glycoproteids, their levels are controlled with the participation of a specific class of the proteolytic enzymes known as matrix metalloproteinases (MMPs) [3]. MMPs in many respects determine activity ofa series ofbiologically active molecules, such as cytokines, inter-leukin (IL-I^), tumor factor of necrosis a (TFN-a), the transforming growth factor of fibroblasts (TGF-^1),
the vascular endothelial growth factor (VEGF) playing a dynamic role in the metabolic processes influencing the cellular proliferation, differentiating migration, apoptosis and angiogenesis. These enzymes provide structural integrity and an elastance of vascular walls. MMPs lead to degradation of the denatured collagen, a fibronectin, laminin, elastin, a vitronectin through their collageno-lytic and elastinolytic effects for the purpose of their utilization and formation of the new, fully functioning molecules.
Gelatinase (MMP-2, MMP-9) are capable to hydro-lyze a fibrillar collagen of the 4th type, and elastase -MMP-7 is produced by vascular cells and inflammatory cells, such as macrophages and polymorphonuclear
neutrophils [1; 2]. The high activity of MMP-2, MMP-9 and MMP-7 is associated with processes of angiogenesis and apoptosis [4]. MMPs expression in physiological conditions is regulated by a series of specific inhibitors which differ on specific action on metalloproteinases. It is established that fabric the inhibitor of metalloproteinases - I (TIMP-1) most actively inhibits MMP-9 while TIMP-2 suppresses activity of MMP-2 [5]. MMP-7 decreases at an expression of TIMP-3 and TGF-^1 [6]. The braking influence TGF-^1 on a catabolism of MMPs sharply strengthens the factor of body height of a connecting tissue raising an expression of TIMP-1 and TIMP-3, the activity of MMP-2 [7; 8] oppresses.
The comparative small molecular mass and solubility in biological liquids provides the ability of MMPs TIMP to get to a vascular blood stream in the quantities proportional to a fabric expression that their definition in blood serum allows. In recent years various MMPs, TIMP and TGF-^1 survey as diagnostic and prognostic markers, change of their activity to normal amounts reflects clinical convalescence [11; 12]. At the same time at the systemic level the nature of activity of MMPs and TIMP at patients with a scleroderma remains almost not studied. It is possible to believe that specification of interrelation of factors of an extracellular matrix and TGF-^1 will help to deepen our ideas of a scleroderma pathogenesis, to define the corresponding tactics of the carried-out treatment of these patients.
The purpose of the this research was to carry out comparative assessment of maintenance of MMP-2, MMP-9 and MMP-7 and also their tissue inhibitors TIMP-1 and TIMP-2 in serum of blood of patients with a systemic and limited scleroderma.
Material and methods of a research
64 patients, including 29 (all women) patients of systemic (SSc) and 35 (26 women and 9 men) of patients with a limited scleroderma (LSc) and also 20 healthy donors similar to age are examined. Average age of patients was 55.3 ± 6.2 years, the average duration of a disease 13.5 ± 4.2 years. Included the analysis of the anamnesis and the objective inspection of patients including a complex of the standard clinical and laboratory methods in inspection (if necessary carried out ultrasonic diagnostics, reovasography, endoscopy, echocardioscopy, an ultrasonic duplex angioscanning). The diagnosis of SSc was verified with use of diagnostic signs, by N. G. Gu-
seva's proposals [13; 14]. The chronic course of a disease took place at 20 (69%) patients, subacute - at 9 (31%). The moderate activity of inflammatory process is noted at 19 (65.5%) patients, minimum - at 10 (34.5%). Clinically SSc was characterized by a multiple syndromes with lesions of various organs, tissues and systems. LSc at all patients was presented by a blyashechny form, to inspis-sation stages at 16 [45; 7] patients, to atrophy stages - at 19 (54.3%). At clinical laboratory and tool inspection of sick LSc of signs of systemacity it wasn't taped.
Content of the researched extracellular matrix enzymes and concentration of TGF-^1 were determined by method of the solid-phase ELISA on the computerized microtablet reader of AT-858 (LTD, China). The MMPs and TIMP level was determined by standard sets for the ELISA: "Human/Mouse/Rat MMP-2 (total)", "Human/Mouse/Rat MMP-9 (total)", "Human MMP-7 (total)", "Human TIMP-1" and "Human TIMP-2" (Quantikine, ROSD Systems, USA), a TGF-^1 - by means of commercial test system (Bender MedSystems, Austria). Analyses were carried out in accordance to vendor's protocol. Concentration of probed indices in blood serum was expressed in nanograms per ml (ng/ml). Statistic data handling was executed on the program Statistica V.7 with determination of average arithmetical index (M) and its error (m). The reliability different was calculated on Student's t-criterion. Correlative communication was calculated according to Pearson (r). Distinctions read statistically authentic in case of significance value p < 0,05.
Results and discussion. The analysis of the received results of researches showed that at sick SC the level of activity of all studied MMPs (MMP-2, MMP-7, MMP-9) was significantly lower, and TIMP (TIMP-1 and TIMP-2) - above, than in control (tab). It is at the same time established what at sick SSc, is more expressed concentration of MMP-2, MMP-7 and MMP-9 - on 17.1 is reduced; 13.9 and 18,3% (P < 0,05), and the TIMP-1 and TIMP-2 level are increased for 18.2 and 19.5% (P < 0,05), in comparison with the data taped at patients with LSc.
Considering an important role ofMMPs and TIMP in a regulation of process of a fibrogenesis, we studied the serumal maintenance of TGF-^1 - a key mediator of a fibrogenesis. It is noticed that TGF-^1 promotes the differentiation of miofibroblast and formation of a fibro-
genic phenotype of fibroblasts, regulates the production platelet a factor of body height (PDGF) stimulates synthesis of components of an extracellular matrix, including collagen and fibronectin [13]. It was established that at sick SC in a systemic blood flow TGF-|1 concentration
Table 1. - The maintenance of MMPs and TIMP of serum of blood at patients of the examined groups
was higher, than in control, including patients SSc groups 98,3% (P < 0,01), with LSc - for 17,1% (P < 0,05). At sick SSc the serumal TGF-|1 level was significantly higher, than at sick LSc for 18,1% (P < 0,05).
The studied indicators, ng/ml Control, n=20 Scleroderma
Systemic, n=29 Limited, n=35
MMP-2 203.62 ± 10.91 140.16 ± 7.71* 168.91 ± 6.92*
MMP-7 4.27 ± 0.15 3.12 ± 0.11*A 3.65 ± 0.11*
MMP-9 310.61 ± 14.95 212.81 ± 8.82*A 260.44 ± 10.68*
TIMP-1 296.54 ± 12.15 422.44 ± 13.51*A 357.32 ± 14.43*
TIMP-2 68.33 ± 3.17 92.73 ± 2.19*A 77.61 ± 2.02*
TGF-|, 90.72 ± 4.35 125.42 ± 4.54*A 106.22 ± 3.72*
Notes: * - P <0,05 in comparison with the control, A - P
To assess the balance of proteolytic and antipro-teolytic activity of serum enzymes in patients with different forms of scleroderma, we determined the index of the ratio of MMP-9/TIMP-1, MMP-2/TIMP-2, MMP-7/TIMP-1 and MMP-7/TIMP-2. It was found that the ratio of MMP-9/TIMP-1, MMP-2/TIMP-2, MMP-7/TIMP-1 and MMP-7/TIMP-2 was significantly lower in SSc group patients than in the control 52.4; 49.3; 71.4 and 45.9% (P < 0.001) respectively, and in patients with LSc - by 30.5; 26.8; 28.6 and 24.6% (P < 0.01), which indicates the dominance of the an-tiproteolytic activity of the MMP regulation enzymes. These processes are significantly higher in patients with SSc compared with those in patients with LSc at 31.5; 30.7% (P < 0.01), 60.0 (P < 0.001), and 28.3% (P < 0.01), respectively, of the observed MMPs/TIMP ratios.
At the same time, we tested the correlation analysis of the relationship between MMPs and TIMP parameters with TGF-|. In SSc patients, the TGF-|1 index had a strong feedback with MMP-2 (r = -0.83, P < 0.001), MMP-7 (r = -0.81, P <0.001) with MMP-9 (r = -0.85, P <0.001) and a direct strong association with TIMP-1 (r = 0.82, P < 0.001), TIMP-2 (r = 0.85; P < 0.001), and in patients with LSc, respectively: r = -0.78; -0.76; -0.79, and -0.77 and 0.76 (P < 0.01), i. e., a strong bond.
Thus, the study showed that in the systemic blood flow of patients with scleroderma, MMPs-MMP-2, MMP-9 and MMP-7 were lower in comparison with con-
<0,05 in comparison with LSc.
trols, against the background of high activity of their inhibitors TIMP-1 and TIMP- 2. Reduction of MMP in the circulating blood of patients with scleroderma is more evidence of inhibition of extracellular matrix remodeling processes, emphasizes the violation of the structural and functional integrity of the skin and the regulation of metabolic processes in the extracellular matrix. Apparently, this is objectively manifested due to their release into the systemic bloodstream. An important factor in reducing the level of MMPs is the activation of their inhibitors TIMP-1 and TIMP-2. It can be assumed that the imbalance in the MMPs/TIMP system is one of the mechanisms for the development of scleroderma. The degree of imbalance in the MMPs/TIMP balance seems to determine the form of scleroderma. In patients with SSc, the degree of MMPs/TIMP is more pronounced, in comparison with patients with LSc. It should be noted that the level of serum TGF-|1 influences the development of imbalance in the MMPs/TIMP system, as evidenced by its correlation with the MMPs/TIMP parameters. The increase in TGF-|1 is associated with a regular increase in the content of inhibitors of MMPs-TIMP-1 and TIMP-2, and inhibition of the activity of proteolysis enzymes-MMP-2, MMP-9 and MMP-7. The revealed disturbances in MMPs activity and their inhibitors can be used for theoretical explanation due to what other mechanisms the processes of fibrogenesis and the role of TGF-|1 in the regulation of the components of
extracellular matrix in patients depending on the form of scleroderma develop.
Conclusions: Consequently, the imbalance between MMPs and their inhibitors, as well as the expression of TGF-^1 in blood serum, play an important role in the breakdown of scleroderma. The degree of these disorders can be considered to determine the
form of scleroderma, it helps to reduce the catabolism of the components of the extracellular matrix and creates a pathophysiological basis for enhancing sclerotic processes in the skin, which should be taken into account in clinical practice to develop a tactic for correcting existing disorders of extracellular matrix function in patients with scleroderma.
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