The Investigation of Strategies to Inhibit Liver Fibrosis Through Modulation of TIMP-1 and -2

Liver fibrosis is a wound healing response characterised by the accumulation of scar tissue (collagen) that follows all forms of chronic liver injury. Progressive fibrosis, which in its end stage causes a gross disruption of the liver architecture (so called ‘cirrhosis‘), impairs liver function and is a major worldwide health problem. Research has shown that liver fibrosis is promoted by proteins (tissue inhibitors of metalloproteinases, TIMPs) which inhibit the enzymes that would otherwise degrade scar tissue. Blockade of TIMP action therefore has considerable potential as an anti-fibrotic strategy. The chief TIMPs in liver fibrosis are TIMP-1 and -2. In the liver, these are produced by hepatic stellate cells (HSC), which importantly, are also the major source of liver scar tissue. We propose to block the action of TIMPs by injecting inhibitory molecules called small inhibitory RNA (siRNA). Specifically, the key objectives of the study will be to:

1. Identify siRNA molecules which successfully inhibit production of TIMP-1 and -2 by HSC.
2. Examine the effect of this on growth and survival of HSC and their production of scar tissue.
3. Investigate the effect of blocking TIMP-1 on recovery from cirrhosis by delivering siRNA targeting TIMP-1 to fibrotic liver, thereby exploring the potential of this approach as a therapy for human liver disease.

Liver fibrosis and its end-stage cirrhosis, represent an increasingly prevalent, major worldwide health problem, which the Chief Medical Officer has identified as a key challenge of the next decade. Individuals usually present at the clinic only once fibrosis is well established and for those with advanced cirrhosis the only current treatment is liver transplantation. However, this approach is limited in most cases and there is a clear and pressing need to develop therapies to combat liver fibrosis. Liver fibrogenesis is promoted by tissue inhibitor of metalloproteinase (TIMP)-1 and -2, which potently inhibit matrix metalloproteinase (MMP) activity, thus protecting extracellular matrix (ECM) from degradation. TIMP-1 also inhibits apoptosis of hepatic stellate cells (HSC), the major cellular source of fibrotic ECM. The aim of the study is to investigate an exciting new anti-fibrotic strategy using small inhibitory RNA (siRNA) to silence hepatic TIMP-1 expression. These studies might ultimately lead to a therapeutic drug for use in humans and will answer key questions regarding the mechanisms underlying the resolution of liver fibrosis. The specific aims of the study will be to:
1. Identify siRNA which effectively silence TIMP-1 and -2 RNA and protein expression in rat and human activated HSC (as assessed by TaqMan PCR of cell lysates and ELISA of culture supernatants), whilst having minimal effect on cell viability or interferon response pathway.
2. Examine the effect of TIMP silencing on HSC phenotype, specifically; (i) apoptosis in response to known apoptogens using fluorescent microscopy, flow cytometry and quantitation of caspase-3 activity; (ii) proliferation by 3H-thymidine incorporation assay; (iii) activation of HSC by Taqman PCR analysis of HSC mRNA for procollagen I, ?-smooth muscle actin (?-SMA), MMP-2 and western blotting for ?-SMA.
3. Establish a dose of lipid-complexed siRNA which effectively silences TIMP-1 in vivo using an acute liver injury model. Expression of TIMP-1, pro-collagen-1 and ?-SMA will be measured in treated and control livers, whilst numbers of activated HSC will be evaluated by immunohistochemistry.
4. Investigate the effect of TIMP-1 siRNA on hepatic fibrosis in a rat model of chronic CCl4 injury/recovery model, specifically:
a. Histological extent of ECM deposition
b. Changes in HSC and other hepatic myofibroblast number by immunostaining and Western blotting of liver homogenates for ?-sma
c. HSC and hepatocyte proliferation and apoptosis using BrdU and TUNEL staining with direct cell counting
d. TIMP and MMP expression by TaqMan PCR of liver homogenates
e. Collagenolytic activity of whole liver homogenates.