Interplay between matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in acute asthma exacerbation and airway remodeling

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1 Egyptian Journal of Chest Diseases and Tuberculosis (2012) 61, The Egyptian Society of Chest Diseases and Tuberculosis Egyptian Journal of Chest Diseases and Tuberculosis ORIGINAL ARTICLE Interplay between matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in acute asthma exacerbation and airway remodeling Ghada Mahmoud Mohamed *, Mohamed Nazmy Farres, Hala Mahmoud Department of Internal Medicine, Ain Shams University Hospitals, Cairo, Egypt Department of Clinical Pathology, Ain Shams University Hospitals, Cairo, Egypt Received 20 April 2012; accepted 30 April 2012 Available online 20 February 2013 KEYWORDS Asthma; MMP-9; TIMP-1; ECM Abstract Background: Airway inflammation and remodeling of extracellular matrix are important features of asthma. Matrix metalloproteinases (MMPs) are group of enzymes expressed in the airways with their inhibitor (tissue inhibitor of MMPs (TIMP) and they are the key responsible for extra cellular matrix (ECM) degradation. Objective: To clarify the role of MMP-9 and TIMP-1 in asthma exacerbation and airway remodeling. Subjects and methods: The study included 3 groups, group A included 22 patients with stable asthma group B included 18 patients during asthma exacerbation and group C of 18 healthy volunteer served as control. All groups were matching age and sex. Levels of MMP-9 and TIMP-1 were measured in the induced sputum of the 3 groups. Serum IgE skin prick test and PEFR were assessed. Results: MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio increased in both A and B groups in comparison to control (P < 0.001). During exacerbation MMP-9 and MMP-9/TIMP-1 ratio showed significant increase for both but TIMP-1 did not show significant change when compared to stable asthmatics. There was significant negative correlation between PEFR and MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio. Conclusion: MMP-9 and TIMP-1 play an important role in pathophysiology of asthma exacerbation and airway remodeling. Clearly, a greater understanding of the pathogenesis of asthma is critical to the development of better therapeutic modalities. ª 2012 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license. * Corresponding author. address: drghmahmoud@gmail.com (G.M. Mohamed). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier ª 2012 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license.

2 36 G.M. Mohamed et al. Introduction Matrix metalloproteinase (MMPs) are family of proteinases with zinc- dependent proteolysis which play important roles in matrix turnover [1]. They are secreted by wide range of stromal and inflammatory cells that are capable of degrading all components of extracellular matrix (ECM) [2]. MMPs, most of which are expressed in the airways, cleave a number of ECM constituents and can be broadly divided into collagenases, gelatinases, stromelysin, elastinases and membrane bound forms [3,4]. Also they play critical roles in lung organogenesis, but their expression, for the most part, is down regulated after generation of alveoli [3]. In particular, MMP-9 (gelatinase B) is reportedly increased in the airways of asthmatic patients [5], are thought to be associated with pathogenesis of airway inflammatory diseases [6,7]. Members of MMPs family are selectively inhibited by tissue inhibitor of MMPs (TIMPs) [8]. Imbalance between MMPs and TIMPs activities has been observed in different pathologic conditions as rheumatoid arthritis, systemic sclerosis and hepatic fibrosis [9]. These data suggested that an excess of MMPs may be responsible for structural degradation of tissue whereas an excess of TIMPs may promote excessive tissue repair and fibrosis [10]. Airway inflammation and remodeling are key histopathologic features in bronchial asthma [11] sever inflammatory cell infiltration into the lung tissue, airway structure changes including epithelium desquamation and abnormal, extracellular matrix (ECM) degradation are observed in the patients with chronic allergic airway diseases [12,13]. These structural changes of the airways are thought to be the result from repeated acute inflammation which is recognized clinically as acute exacerbation [14]. However, the relationship between airway remodeling and inflammation is poorly understood [13]. Recent studies have suggested that episodes of acute inflammation promote airway remodeling by altering the homeostasis of ECM components [13]. It was suggested that MMPs have been implicated in the turnover and airway remodeling of ECM. Consequently, MMPs and TIMPs and more specially the balance between these two biological components may have a crucial role in the pathophysiology of asthma exacerbation and subsequent airway remodeling [15]. The aim of the present work is to clarify the role of MMP-9 and TIMP-1 in asthma exacerbation and airway remodeling. Subjects and methods This study included 40 atopic (atopy defined as having history of atopy and positive skin test) asthmatic patients (diagnosed according to British thoracic society 1997) [16]. They were selected from Allergy and Immunology outpatient clinic of Ain Shams University Hospitals, Cairo, Egypt, in the duration from February 2011 to July They were divided into 3 groups Group A Twenty two patients with stable asthma, they were 11 females and 11 males with mean age 34 ± 10.5 years. Their symptoms and PEFR were stable with no changes in the treatment for at least 1 month [17]. Group B Eighteen patients were recruited during acute exacerbation. They were 9 females and 9 males with mean age 33 ± 10.4 years. Exacerbation was diagnosed according to breathlessness, audible wheezes on auscultation and morning peak expiratory flow rate (PEFR) <70% of the personal best value in the last 3 months [17,18]. Group C Third group of 18 healthy volunteer subjects served as control group. They were 10 females and 8 males within mean age 32.2 ± All groups were matching age and sex. Exclusion criteria Patients with concomitant acute illnesses or pneumonia, patients receiving corticosteroids during the last month and patients with other medical diseases were excluded from the study. Also, current smokers or ex-smokers were excluded. All participants were subjected to the following after written consents: (1) Full medical history and clinical examination. (2) Skin prick test. Epicutaneous prick method using standard allergen extracts (mite, mould, grass, mixed pollens and house dust) negative control (saline) and positive control (histamine). All allergen extracts were prepared in Ain-Shams allergy and Immunology Extract unit by aqueous vaccine method (weight/volume) method (1/10 dilution) according to Neuman and Arman 1988 [19] positive skin test was defined as a wheal 3 mm in diameter greater than the wheal of negative control. (3) Serum total immunoglobulin E (IgE): in Iu/L by ELISA. Quantitive measurement by a commercially available ELI- SA kit. (Med Biotech, Inc., Agenzme company, Industrial Road, San Carlos, CA, USA). (4) Peak expiratory flow rate (PEFR): Using traditional peak Flow meter. Results were expressed as percentage of the predicted values considering the height, age and sex. (5) Sputum induction and measurement of both MMP-9 and TIMP-1 (by ELISA technique from oncogen TM researches products, sandwich enzyme) Result were expressed in ng/ml [20]. Sputum induction According to Kanazawa [21] and his colleagues, patients were premedicated with inhaled salbutamol, after washing their mouths, they inhale normal saline by nebulizer and encouraged to cough deeply after 5 and 10 min into polypropylene containers.

3 Interplay between matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 37 Statistical analysis The descriptive statistics (mea, standard deviation, minimum and maximum values) for all groups were calculated. Wilcoxon rank-sum test was used for two groups. Spearman s rank analysis used to evaluated the correlation between data. Results Group A: (stable asthmatics) showed high significant increase in IgE, PEFR, MMP-9, TIMP-1 and MMP-9/ TIMP-1 ratio when compared to control group C (P < 0.001) (Table 1). Group B: (asthmatic patients during exacerbation) showed high significant increase in IgE, PEFR, MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio when compared to control group C (P < 0.001) (Table 2). Group A: (stable asthmatic) showed high significant increase in IgE, MMp-9 and MMP-9/TIMP-1 ratio when compared to group B (asthmatic during exacerbation) (P < 0.01), (P < 0.001), (P < 0.001) respectively. PEFR showed much reduction (P < 0.001). TIMP did not show significant change during exacerbation (Table 3). Among the patients of group A, there was negative significant correlation between PEFR and MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio. (Table 4). Among the patients of group B, there was negative significant correlation between PEFR andmmp-9, TIMP-1 and MMP-9/TIMP-1 ratio (Table 5). Discussion Airway remolding is a major change responsible for irreversible asthmatic air flow restriction [6]. MMP-9 and TIMP-1 are thought to be deeply involved in the pathogenesis of chronic airway disease [14]. Upregulation of MMP-9 has been previously reported in airways with chronic inflammation and remodeling such as chronic obstructive pulmonary disease including emphysema, pulmonary fibrosis [22,23]. Although the mechanism is still under investigation and not accurately known, the imbalance between MMP-9 and TIMP-1 is considered a major theory to explain the progression of asthmatic airway remodeling [6]. Results of the present work showed that asthmatic patients had increased levels of MMP-9 and TIMP-1. It is increasingly accepted that airway inflammation and remodeling which oc- Table 1 Comparison between group (A) (stable asthma) and group (C) (control group). Parameter Group A Group C Z P Sig Age (years) 34.9 ± ± >0.05 NS PEFR% 79.2 ± ± <0.001 HS IgE: IU/L 483 ± ± <0.001 HS MMP-9 (ng/ml) 166 ± ± <0.001 HS TIMP (ng/ml) 339 ± ± <0.001 HS Ratio 0.48 ± ± <0.001 HS Table 2 Comparison between group (B) (asthma exacerbation) and group (C) control group. Parameter Group B Group C Z P Sig Age (years) 33.6 ± ± >0.05 NS PEFR% 62.6 ± ± <0.001 HS IgE: IU/L 759 ± ± <0.001 HS MMP-9 (ng/ml) 311 ± ± <0.001 HS TIMP (ng/ml) 381 ± ± <0.001 HS Ratio 0.79 ± ± <0.001 HS Table 3 Comparison between group (A) and group (B). Parameter Group A Group B Z P Sig Age (years) 34.9 ± ± >0.05 NS PEFR% 79.2 ± ± <0.001 HS IgE: IU/L 483 ± ± <0.001 HS MMP-9 (ng/ml) 166 ± ± <0.001 HS TIMP (ng/ml) 339 ± ± >0.05 NS Ratio 0.48 ± ± <0.001 HS

4 38 G.M. Mohamed et al. Table 4 Correlation between MMP-9, TIMP and ratio and all studied parameters among group A. Parameter R MMP-9 Sig TIMP-1 Sig Ratio Sig TIMP Sig Ratio Sig NS Age NS NS NS PEFR Sig Sig Sig IgE NS Ns NS Table 5 Correlation between MMP-9, TIMP and ratio and all studied parameters among group B. Parameter R MMP-9 Sig TIMP-1 Sig Ratio Sig TIMP Sig Ratio Sig NS Age NS NS NS PEFR Sig Sig Sig IgE NS Ns NS cur in asthma can cause functional alteration because of quantitative changes in airway wall compartments or by changes in the biochemical composition of the various constituents of the airway wall [24]. This goes with a previous workers who suggested the involvement of MMP-9 in the airway remodeling process in asthma. Upregulated expression of MMPs are thought to mainly contribute to the abnormal degradation of ECM in the airways, which lead to the physiological structural changes of the airways and is also thought to be one of the main reasons of airway hyper responsiveness [25]. The current work denoted that TIMP-1 level was significantly higher in asthmatics when compared to control. This supported the results of previous work which suggested that the increased level of TIMP-1 in asthmatic patients may represent an endogenous protective mechanism to down regulate the proteolytic activity of MMPs in the lung parenchyma. Increased concentration of TIMP-1 in asthma can be the result of several mediators released during development of airway inflammation as transforming growth factor-b [26]. Our study showed increased MMP-9/TIMP-1 ratio in asthmatics. This results supported by results of previous workers who attributed this high ratio to predominance of inflammation over fibrosis [22,27]. Moreover, another work reported low MMP- 9/TIMP-1 ratio in sever asthmatics with poor response to steroids due to excessive fibrosis [20]. The results presented in our study demonstrated significant increase in MMP-9 but no significant change in TIMP-1 in asthmatics during exacerbation when compared to stable asthmatics. These results were in agreement with another worker who found that asthma exacerbation promote airway remodeling by altering MMP-9 mediated ECM homeostasis [28]. It was reported that levels of MMP-9 not TIMP-1 increase after inhaled allergen challenge in sputum of allergic asthmatic patients [29]. Our results revealed significant negative correlation between MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio with PEFR. i.e. more airway obstraction was accompanied by more release of MMP-9 and TIMP-1. This goes with a previous work which showed that depletion of MMP-9 also attenuated the peribronchial fibrosis and airway hyper-responsiveness induced by chronic antigen exposure [30]. Also we found that there was no significant correlation between MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio and age and IgE. These results disagree with previous workers who suggested that IgE trigger reactions to inhaled allergens leading to allergic inflammation and release of MMP-9 [31]. The lack of correlation to IgE in the present study may be because acute exacerbation is due to multi-factors including allergen exposure. Conclusion Our results suggested that the processes of airway inflammation and remodeling are coordinated, and MMP-9 and TIMP-1 are cooperative in these pathophysiological changes of allergic airway disease. These findings might provide a better understanding of the regulation of airway inflammation and remodeling in allergic airway disease. MMP-9 may be a potential target for management of exacerbation of asthma and prevention of subsequent airway remodeling. References [1] P.K. Jeffery, A. Laitinen, P. Venge, Biopsy markers of airway inflammation and remodeling, Respiratory Medicine 94 (2000) [2] D. Mosher, J. Sottile, J. McDonald, Assembly of extracellular matrix, Current Opinion in Cell Biology 4 (1992) [3] K.J. Greenlee, Z. Werb, F. Kheradmand, Matrix mettalloproteinases in lung: multiple, multifarious, and multifaceted, Physiological Reviews 87 (1) (2007) [4] V. Lagente, B. Manoury, S. Nénan, et al., Role of matrix metalloproteinases in the development of airway inflammation and remodeling, Brazilian Journal of Medical and Biological Research 38 (10) (2005) [5] F.W.S. Ko, C. Diba, M. Roth, et al., A comparison of airway and serum matrix metalloproteinase-9 activity among normal

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