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Role of matrix metalloproteinase-9 in cystic fibrosis lung disease and evaluation of therapeutic strategies

Wagner, Claudius

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Abstract

Current hypotheses suggest that a disrupted antiprotease-protease balance results in structural tissue damage in cystic fibrosis (CF) lung disease. Clinical studies identified elevated neutrophil elastase (NE) levels as risk factor for the onset of structural lung damage in young CF patients. A recent study reported that levels of NE-activated matrix metalloproteinase (MMP)-9 correlated with the progression of bronchiectasis in CF patients. Studies in mice with lung-specific overexpression of the β subunit of the epithelial Na+ channel (βENaC-Tg) confirmed a crucial contribution of NE activity to CF-like lung disease. However, NE deletion only partially reduced structural lung damage, i.e. emphysema, suggesting that additional factors contribute to tissue destruction in βENaC-Tg mice. The present study investigates the pathogenic role of MMP-9 in βENaC-Tg mice. Similar to CF patients, MMP-9 protein levels were elevated in bronchoalveolar lavage (BAL) fluid of βENaC-Tg mice as detected by gelatin zymography. To determine the contribution of MMP-9 to the pathogenesis of βENaC-Tg mice, mortality, pulmonary inflammation, transcript levels of mucins, distal airspace enlargement and soluble MMP-9 activity in BAL fluid were studied in WT, Mmp9-/-, βENaC-Tg and βENaC-Tg/Mmp9-/- mice. Additionally, measurement of lung function in NE or MMP-9 deficient βENaC-Tg mice enabled the comparison of the individual influence of the respective protease on lung tissue mechanics. Inflammatory cytokines and neutrophil chemoattractants were elevated in βENaC-Tg mice and not altered in BAL fluid of βENaC-Tg/Mmp9-/- mice. In addition, pulmonary leukocyte infiltration, mucin expression and goblet cell metaplasia were not dependent on MMP-9 and elevated to similar levels in βENaC-Tg and βENaC-Tg/Mmp9-/- mice. Lung volume measurement and morphometric quantification of distal lung histology showed an increased emphysema severity in βENaC-Tg mice which was not reduced in βENaC-Tg/Mmp9-/- mice. Free soluble MMP-9 activity could not be detected in BAL fluid neither of βENaC-Tg mice nor of littermate controls. Increased concentrations of tissue inhibitor of matrix metalloproteinases (TIMP)-1 as main endogenous MMP-9 inhibitor indicate an intact antiprotease-protease balance in BAL fluid of βENaC-Tg mice. Lung function testing revealed no improvement of static compliance, inspiratory capacity or tissue elastance by Mmp9 deletion in βENaC-Tg mice. The assessment of lung function displayed a significant amelioration of these parameters in βENaC-Tg/NE-/- compared to βENaC-Tg mice. According to these results, preclinical trials were performed targeting NE with the small molecule inhibitor sivelestat to test the effect on disease development in newborn βENaC-Tg mice. Systemic sivelestat delivery by intraperitoneal injections reduced airway mucus obstruction but did not affect leukocyte infiltration or emphysema severity in two week-old βENaC-Tg mice compared to vehicle controls. In summary, the analysis of MMP-9 deficient mice suggests that MMP-9 is not crucial factor in the pathogenesis of βENaC-Tg mice. This may be related to balanced antiprotease levels in the lungs of βENaC-Tg mice, which are frequently depleted in CF patients. Therefore, it is difficult to deduce from the current studies the pathogenic potential of MMP-9 in more severe stages of CF lung disease. In contrast to Mmp9 deletion, NE deficiency improved lung function in βENaC-Tg mice. The preclinical trials with sivelestat showed that NE inhibition was insufficient to reproduce the results of NE deletion in βENaC-Tg mice. Thus, further treatment studies are needed using compounds with improved pharmacokinetic properties that enable effective NE inhibition in pulmonary tissue.

Item Type: Dissertation
Supervisor: Wölfl, Prof. Dr. Stefan
Date of thesis defense: 13 June 2019
Date Deposited: 26 Jun 2019 12:10
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
610 Medical sciences Medicine
Controlled Keywords: cystic fibrosis, protease, lung disease
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