MICA: Illuminating mechanisms regulating the birth, life and death of the myofibroblast to inform the development of antifibrotics for liver disease.

The ability of our bodily tissues to effectively repair cellular damage caused by environmental agents (viruses, bacteria, toxins, dietary factors etc) is critical for prevention of further damage or infection and for the healthy restoration of organ function. Key to tissue repair is the formation of temporary scars and contraction of wounds in order to "wall off" sites of damage while tissue healing takes place. If tissues suffer repeated damage over extended periods of time then scars are modified such that they are more difficult to break down during healing and they can spread to affect other parts of the organ. This is known as "fibrosis" and it can progress to the stage where the normal architecture and function of an organ is transformed to such an extent that it fails and the only current treatment options are transplantation or palliative care. Ageing appears to reduce the ability of organs to repair normally and as a result fibrosis is becoming more common in the general population, with recent estimates suggesting it underlies up to 40% of all deaths in the developed world. At present we have no proven effective therapies or preventative treatments for tissue fibrosis in any organ system. The liver is the organ responsible for detoxification and plays a major role in clearance of pathogens, however cells in the liver are equally susceptible to damage and infection as those in other parts of the body. As a consequence the liver is particularly susceptible to fibrosis where toxins or pathogens persist, and as we age when the regenerative capacity of the liver begins to falter. Chronic liver disease is currently the only common cause of death that is on the rise in the UK, with 16,000 deaths reported in 2008 and worryingly the trend is towards disease developing in people in their middle-life years. An effective antifibrotic that halts the development of scars and/or that helps break down existing scars in the liver would have a major impact on morbidity and mortality in patients with chronic liver disease.

The research proposed in this application for a 5-year grant will build on work in the laboratory of the PI and his team that has identified a specialised cell in the diseased liver that promotes the formation, maintenance and spread of scars. This cell is known as a liver myofibroblast and it is generated chiefly by alterations in the properties and behaviour of liver cells that normally function to store Vitamin A. Upon injury or infection these so called "hepatic stellate cells" transform into myofibroblasts that produce vast quantities of scar tissue and are able to proliferate and migrate to cause fibrosis in the liver. The PI has been part of an international research effort that has confirmed that manipulating hepatic stellate cell-derived myofibroblasts can halt and even reverse fibrosis. The challenge for the next 5 years that will be tackled by the research team is to illuminate biochemical signals that regulate myofibroblast fate and function with the aim of finding biochemical "Achilles heels" that can be manipulated to stop or even hopefully reverse fibrosis. The research will use a combination of human cell culture, relevant mouse disease model systems and diseased human liver tissues to identify which signals can be manipulated to bring about antifibrotic effects. The team will use this knowledge to look for either existing drugs or new compounds (by chemical library screening) to find safe and effective treatments. This ambitious goal will be facilitated by a strong working partnership between the research team and the global biopharmaceutical company GlaxoSmithKline (GSK), the latter having a dedicated team of 40+ scientists working on discovery and development of antifibrotics at its Stevenage site. GSK will lend support with manpower, access to specialised scientific knowhow and provision of compounds and research tools as well as providing routes for translation to the clinic.

The research will employ the following methodologies in order to meet the stated aims and objectives in the "Case for Support".

1. Human primary liver myobroblasts. These cells will be used to determine mechanisms by which epigenetic and transcription pathways influence fibrogenesis. The cells will be isolated from the normal margins of liver tissue resected for the removal of secondary tumours provided by liver surgeons (Profs White and Mannas) at the Newcastle NHS Trust Freeman Hospital. Isolated cells will be purified by density centrifugation and cultured in serum-containing media.

2. A human myofibroblast cell line, LX2, will be used where high numbers of myofibroblasts are required for detailed studies such as chromatin immunoprecipitation, or where there is a need to modulate the expression of genes under study by transfection-based approaches. The LX2 line was generated in the laboratory of Prof Scott Friedman and is highly representative of the fibrogenic phenotype of primary liver myofibroblasts.

3. Archived paraffin-embedded diseased human liver tissue will be used for immunohistochemical detection of key regulatory proteins identified during the research as important to validate in human diseased tissue. The tissue is managed by the Newcastle University and NHS Trust Biobank and will be supplied as anonymised liver slide sections, with the original tissue blocks being retained by the Biobank.

4. Genetically modified mice (C57/Bl6) for key fibrogenic regulators such as MeCP2 and JunD will be used for supply of myofibroblasts in which these genes are either deleted or mutated into functionally attenuated forms, these studies enabling gene function to be examined with a high degree of confidence. The mice will also be employed in animal models of liver disease.

The main beneficiaries from the research will be:

1. General public stakeholders associated with the MRC strategic themes of "Ageing" and "Lifelong Health and Wellbeing".
Fibrosis can occur in almost every soft tissue organ and impacts directly on fitness/vitality or in the worst cases leads to organ failure. Estimates suggest that 40% of all deaths in the developed world have fibrosis as an underlying contributory pathology. Although fibrosis is seen in young adults (in particular for liver disease) it is an age-associated pathology. Furthermore, the age of onset of chronic disease is a strong prognostic indicator for progression to fibrosis. With the average lifespan of UK people increasing each year fibrotic disease and its impact on quality of life and economic burden will be substantive. There is therefore an unmet need for drugs that will prevent progressive fibrosis which would benefit thousands of UK patients. With previous MRC funding the PI and the research team have delivered discoveries that have translated into (i) An EME-funded multi-centre clinical trial led by Prof Day (Newcastle) assessing the efficacy of losartan in non-alcoholic fatty liver disease (ii) Identification of 5-HT2B antagonists as antifibrotics in the liver expected to lead into clinical studies. It is anticipated that with continued MRC support and through partnership with GSK, the Mann laboratory will make further discoveries for clinical translation and ultimately leading to safe and effective antifibrotics.

2. Academic/private sector collaboration and future benefits for economy.
An effective antifibrotic will create a multi-billion pound worldwide market. Fibrosis is therefore now a key disease theme in the pharmaceutical and biotech industry, as evidenced by the recent establishment of a GSK fibrosis DPU at Stevenage. Mann and Oakley lead a Newcastle partnership with GSK to deliver antifibrotics in liver and kidney disease. Two of the major GSK drug leads (under disclosure agreements) in development emerged directly from MRC-funded work in the Mann lab carried out in the past 3 years. Mann also has PhD studentships for fibrosis-related projects with two biotech companies (Medimmune and UCB) which are helping to inform these companies on their future strategic investment in fibrosis. There is therefore strong evidence for a significant competitive advantage for UK industry in having first exposure to new fibrosis drug targets emerging from the high quality MRC-funded research emerging from the Mann laboratory.

3. International academic colleagues working in chronic disease and fibrosis.
The PI is widely considered to be a world leader in the emerging field of tissue fibrosis. Published discoveries, international conference presentations of work on mechanisms of liver fibrosis and provision of research tools and concepts from the Mann lab have combined to stimulate research across the globe to find evidence of similar fibrogenic mechanisms in other organ systems (lung, skin, kidney and eye). This has led to the identification of core fibrogenic targets for drug discovery and has raised the worldwide academic profile of fibrosis research. This impact is expected to expand if the Newcastle team are funded with their first programme-level grant.

4. Postdoctoral Scientists funded on the grant.
The PI has mentored the career development of three postdoctoral scientists (Oakley, J Mann and Prof Matt Wright) to senior academic posts and each is now emerging as international leaders in the fibrosis field. The Mann lab is currently comprised of trainee researchers from the UK, Spain, Australia, India, Turkey, Germany and past lab members now hold postdoctoral or industry posts in the USA, Australia and Europe. The lab is therefore a high quality environment for training and career development and this will be of direct benefit to postdoctoral and PhD student scientists working on the research programme.