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Title: An investigation of the effect of tissue inhibitors of metalloproteinases-1 and -2 on hepatic stellate cell survival
Author: Murphy, Francis Robert
ISNI:       0000 0001 3433 4672
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2003
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The aim of this study was to determine the effect of TIMP-1 and TIMP-2 on HSC survival and determine whether this effect was through effects on inhibition of MMP activity. In vivo studies of experimental liver fibrosis demonstrated a correlation between resolution of fibrosis, falling, TIMP-1 mRNA, loss of HSC and transient increase in collagenolytic activity. This data suggested that TIMP-1 was likely to have an effect promoting HSC survival. In vitro studies of culture activated HSC demonstrated that neither TIMP-1 nor TIMP-2 regulated HSC proliferation suggesting that the pro survival effect was likely to be via inhibition of apoptosis. Incubation with TIMP-1 or TIMP-2 significantly reduced apoptosis of HSC induced by a number of stimuli in a dose-dependent manner. Neutralising antibodies to TIMP-1 and TIMP-2 increased HSC apoptosis compared non-immune IgG control. Whilst both a synthetic selective inhibitor of MMP-2 and a broad spectrum synthetic MMP inhibitor reduced HSC apoptosis, a non-functional mutated TIMP-1 (T2G mutant) had no effect indicating that the inhibition of apoptosis by TIMP-1 was via inhibition of MMP activity. MMP-2 like TIMP-1 is expressed by activated HSC. Addition of recombinant active MMP-2 to HSC resulted in significantly enhanced apoptosis and was associated with cleavage of N-cadherin which could be reduced by co-incubation with recombinant TIMP-1 but not by the non-functional T2G mutant TIMP-1. Furthermore, selective MMP-2 inhibitors protected N-cadherin from cleavage. Active MMP-2 cleaves intact N-cadherin into similar sized fragments in vitro. Blockade of N-cadherin binding with blocking antibody or HAVDI blocking peptide promoted HSC apoptosis, suggesting N-cadherin is a potential target mechanism for the regulation of survival and apoptosis of HSC via active MMP-2 mediated cleavage. Finally, to determine if effects on apoptosis by TIMP-1 were through changes in cell-matrix interactions, HSC plated onto a mutated collagen that is resistant to degradation by MMP-2 were more resistant to apoptosis induced by active MMP-2 than HSCs plated onto normal wild type collagen-1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available