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Title: The activation of lung fibroblasts by human mast cell tryptase : a role for protease activated receptor-2 (PAR-2)
Author: Akers, Ian Arthur
ISNI:       0000 0001 3403 6747
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2001
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One of the features of asthma is the remodelling of the airway wall with increased deposition of extracellular matrix molecules, in particular collagen and proteoglycans, in the lamina reticularis beneath the epithelial basement membrane. The mechanism of this deposition is currently unknown and current therapies have little effect on this aspect of asthma pathology. Fibroblasts and myofibroblasts, which are thought to be major producers of extracellular matrix molecules, are present within the lamina reticularis, however the mechanisms by which these cells are activated are unclear. Within the asthmatic airway there are also increased numbers of activated mast cells that produce a range of mediators capable of modulating fibroblast function. One such mediator, tryptase, is a known fibroblast mitogen and profibrotic agent. The mechanism by which this occurs is also unclear, although recent evidence suggests that tryptase is able to activate protease activated receptor (PAR)-2, a member of a family of 7-transmembrane, G-protein coupled receptors activated by enzymatic cleavage. The overall aim of this thesis was to examine fibroblast mitogenesis and extracellular matrix production in relation to airway remodelling associated with asthma. In doing so, I addressed the hypothesis that mast cell tryptase modulates fibroblast function by the activation of PAR-2 present on the cell surface. To address this hypothesis the effects of tryptase, trypsin, a known activator of PAR-2, and small polypeptide activators of PAR-2, SLIGKV and SLIGRL, were examined on fibroblast proliferation, procollagen and proteoglycan metabolism. All four agonists induced lung parenchymal and airway fibroblast proliferation. The two enzymes were more potent than the two activating peptides. The mitogenic responses of the enzymes were dependent of their catalytic activity. It was found that lung parenchymal and airway fibroblasts expressed PAR-2 mRNA, using reverse transcriptase polymerase chain reaction, and PAR-2 protein was localised to the cell surface by immunohistochemistry using polyclonal antibodies. The same antibodies inhibited trypsin- and partially inhibited tryptase-induced fibroblast proliferation, demonstrating that both enzymes activated PAR-2. It was unclear whether PAR-2 was present on fibroblasts in the airway. Therefore using PAR-2 antibodies and a cell cytoskeletal marker for smooth muscle F-actin, phalloidin, it was possible to co-localise PAR-2 and actin filaments suggesting that PAR-2 is expressed on myofibroblast-like cells in human bronchus. Finally, results using a procollagen α2(I) promoter linked to a luciferase reporter gene demonstrated that both tryptase and the PAR-2 activating peptides were able to stimulate procollagen gene promoter activity. However, neither agonist induced increases in the steady-state levels of α1(I)-procollagen mRNA or procollagen protein metabolism assessed by measurement of hydroxyproline. This inconsistency may be due to known effect of tryptase to induced the release, and activate, matrix metalloproteinases that are capable of degrading newly formed and/or deposited procollagen. Tryptase and trypsin had variable effects on large and small molecular weight proteoglycan metabolism in human lung fibroblast cell cultures. The data presented in this thesis demonstrate that tryptase mediates its effects on fibroblast mitogenesis, at least partly via the activation of PAR-2 and is consistent with the hypothesis that tryptase may play an important role in fibroblast proliferation and matrix deposition in the airways of asthmatic patients.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Asthma