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Title: The investigation of strategies to inhibit liver fibrosis by targeting tissue inhibitor of metalloproteinases with RNA interference
Author: Fowell, Andrew
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2010
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Current evidence suggests that tissue inhibitor of metalloproteinase (TIMP)-l and -2 are expressed by hepatic stellate cells (HSC during the course of their activation to a profibrotic myofibroblastic phenotype and play an important role in liver fibrogenesis. Therefore, inhibition of these key molecules represents an attractive strategy for treatment of liver fibrosis. RNA interference (RNAi) is a naturally occurring cellular mechanism involving sequence-dependent silencing of target gene expression througn degradation of messenger RNA {m RNA). Evidence has rapidly emerged that components of this native mechanism such as short interfering RNA (siRNA) and microRNA (miRNA) may be harnessed therapeutically. It was hypothesised that inhibition of hepatic stellate cell TIMP-l and -2 expression by RNA interference has an antifibrotic: effect both in vitro and in vivo. The aims of the study were to: i) Identify siRNA which effectively silence TlMP-l and -2 expression in rat activated HSC; ii) Examine the effect of TIMP silendng on HSC phenotype, in particular apoptosis and proliferation; Hi) Investigate the role of endogenous RNA intenernce acting via miRNA in the regulation of TIMP-l and -2 expression by HSC; iv} Using an acute liver injury model, establish a clinically relevant means of delivering siRNA l miRNA which effectively silences TIMP-l in vivo and if successful, apply this to a chronic model of liver fibrosis and recovery. Culture activated primary rat HSC were transfected with TIMp·1 and 2siRNA by elec.tloporal.ion and target mRNA and protein expression .determined. The effect of TIMP silencing on HSC MMP-2 inhibition, proliferation and apoptosis was examined. Global miRNA expression during MSC activation was examined and "the potential role of candidate miRNAs in HSC TIMP expression. proliferation and apoptosis studied by miRNA overexpression and inhibition. Finally, the efficacy of TIMP-l siRHA was tested in vivo using peripheral liposomal delivery in a ca~ model of acute liver injury TIMP-l and -2 siRNA elec:rroporation were highly effective means of silencing HSC llMP expression in vitro. Silencing ofllMP-l or -2 removed a functional MMP suppressive effect in HSC cultures but did not affect HSC apoptosis in response to serum·deprivation. TIMP-l silencing inhibid HSC pro1iferation and was associated with reduced Akt phosphorylation, suggesting an autocrine role for TIMP· 1 in enhancing proliferation in fibrosis. activation of rat HSC was accompanied by marked up- and down-regulation of multiple miRNAs miR-143 was markedly unregulated with HSC activation and functional studies Suggested a profibrotic role for this miRNA in HSC Acute CCI, injury in rats increased hepatic TIMP-l expression but this was not attenuated byllMP-l siRNA delivered peripherany under normal pressures using a liposomal vector., perhaps due to preferential uptake by resident liver macrophages. In conclusion, these data shed new light on the role of TIMP-I and of miRNAs in HSC function. The efficacy of a siRNA-mediated anti-TIMP-l strategy has been shown in vitro, although the mechanisms of efficient delivery of reagents capable of targeting hepatic TIMP-l expression in vivo await further elucidation.
Supervisor: Collins, Jane Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RC Internal medicine