Myofibroblast differentiation in idiopathic pulmonary fibrosis: epigenetic mechanisms and therapeutic targeting by cAMP

Lead Research Organisation: University of Nottingham
Department Name: School of Medicine

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive and devastating lung disorder characterised by irreversible scarring of the lung. IPF affects over 8,000 people in the UK. It causes disabling breathlessness, and often leads to death within a few years. The cause of the disease is unknown despite much research and there is no effective therapy to stop or slow the disease progression. Myofibroblasts (a cell type) are the major cells responsible for the scar formation (fibrosis) and can be transformed from fibroblasts and epithelial cells (two other cell types) by the potent pro-fibrotic mediator termed transforming growth factor beta1 (TGFbeta1). Under normal conditions, the formation of myofibroblasts is inhibited by a potent antifibrotic mediator termed prostaglandin E2 (PGE2). However, during the process of myofibroblast formation, the cells gradually lose the capability to produce this mediator, resulting in excessive fibroblast growth and scar formation. Our preliminary studies suggest that exogenously supplied PGE2 and related drugs can inhibit and, to some extent, reverse the TGFbeta1-induced transformation from normal lung fibroblasts and epithelial cells to myofibroblasts, thus could stop the excessive scar formation and be beneficial for patients with IPF as novel therapies. In the studies proposed here we will use normal lung fibroblasts and epithelial cells to study the mechanisms of TGFbeta1-induced transformation to myofibroblasts and the effect of exogenous PGE2 and related drugs on the transformation and to determine the molecular mechanisms of action of these drugs. In addition, we will also use lung tissues from patients with IPF and a mouse model of pulmonary fibrosis to confirm the findings with cell studies. We believe that the outcome of our studies will improve our understanding of the pathogenesis of IPF and provide preclinical evidence for PGE2 and related drugs as potential novel therapies to benefit IPF patients in the near future.

Technical Summary

Idiopathic pulmonary fibrosis (IPF) is a deadly respiratory disease of unknown aetiology with no effective therapy. IPF is characterised by fibroblast accumulation and activation and excessive collagen deposition, leading to distortion of the alveolar architecture, progressive loss of lung function, and ultimately death. Myofibroblasts are the primary effector cells in IPF and can be differentiated from lung fibroblasts and epithelial cells. PGE2, a major prostanoid generated by cyclooxygenase-2 (COX-2) from lung fibroblasts and epithelial cells, inhibits myofibroblast differentiation through cAMP. However, PGE2 is reduced due to COX-2 repression in this process, leading to unopposed myofibroblast differentiation. Increasing evidence suggests that epigenetic regulation plays a key role in myofibroblast differentiation and that cAMP elevating agents can compensate for exogenous PGE2 loss to prevent and even reverse myofibroblast differentiation.
We hypothesize that lung fibroblasts and epithelial cells are epigenetically reprogramed during their differentiation to myofibroblasts in IPF, resulting in gene specific epigenetic modifications relative to the expression and repression key genes and that cAMP elevating agents can prevent, even reverse, the differentiation at least partly through epigenetic regulation. We aim to elucidate the epigenetic mechanisms and cAMP-mediated inhibition of myofibroblast differentiation by studying TGF-beta1-induced fibroblast-to-myofibrobalst differentiation and epithelial-mesenchymal transition in vitro, validating key epigenetic findings relative to gene expression in IPF lung tissue and in a mouse model of pulmonary fibrosis, and evaluating the anti-fibrotic effect of cAMP elevating agents and epigenetic inhibitors in the mouse model. The outcome of this project will improve our understanding of IPF pathogenesis and provide preclinical evidence for these agents as potential novel therapies to benefit IPF patients.

Planned Impact

In addition to the academic beneficiaries described separately, this research has the potential to benefit a wide group of people. The group of people that are most likely to benefit will be patients with idiopathic pulmonary fibrosis (IPF). IPF is a chronic fatal lung disease of unknown aetiology and with no effective treatment and a median survival of 2.4 years. This study aims to develop novel therapeutic strategies based on a better understanding of IPF pathogenesis. Successful delivery of this study will not only improve the understanding of IPF pathogenesis but also provide pre-clinical evidence for cAMP elevating agents and epigenetic inhibitors as potential new therapies for IPF patients. The drugs we plan to test are the PDE4 inhibitors roflumilast and the HDAC inhibitor LBH589, which are in clinical use for COPD and cancer treatment, respectively, but their use in IPF treatment has not been tested. We have preliminary evidence that the drugs have potent anti-fibrotic effect from in vitro studies and we are going to test their effect in a murine model of pulmonary fibrosis. Positive results can soon be translated into clinical trial either locally through the Nottingham Respiratory Research Unit or by clinicians in the UK and abroad. There are approximately 5000 new cases of IPF in the UK each year, which represents a huge burden for NHS. There is a realistic prospect that our results can lead to new therapies to improve the quality of life of IPF patients and reduce the cost of caring, and thus will have a big positive impact on the nation's health and wealth.
The public sector will benefit through the development of new scientific knowledge, and the enhancement of the research experience and skills of the researchers involved. The post-doctoral research fellows working on the project will also develop research and professional skills, such as writing, inter-people communication and presentation, which they can apply in both academic and non-academic sectors.
Results from the research, particularly the drug effect, will likely to attract the interest and investment of pharmaceutical companies to redesign and test more effective and less toxic drugs for IPF treatment, which in turn will benefit them for their research knowledge and skills and product output in the long run and benefit the economy of the country.

Publications

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Coward WR (2018) Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis. in American journal of respiratory cell and molecular biology

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Coward WR (2014) A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

 
Description Editor in Chief Thorax
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact As Editor-in-Chief of Thorax (the official journal of the British Thoracic Society and 4th highest Respiratory Journal) we have completely revised the instructions for authors to emphasise scientific rigour with explicit guidelines for research of many disciplines.
URL http://thorax.bmj.com/pages/authors/
 
Description Overseas research visit
Amount £1,860 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 05/2016 
End 06/2016
 
Description Visiting Fellowship
Amount £6,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 05/2014 
End 08/2014
 
Title Murine precision cut lung slice (PCLS) in vitro lung fibrosis model 
Description Precision cut lung slices (PCLS) maintained in vitro provide a valuable bridge between studies in isolated cells and whole animals and can be very useful for elucidating multifaceted mechanisms of pulmonary vascular diseases. They are advantageous over in vivo and single-cell culture systems: 1) maintain near identical anatomy to in vivo tissue and preserve resident cell-cell and cell-matrix interactions, allowing assessment of cellular responses in a native tissue environment; 2) focus analysis on resident lung cells without complicating influences of recruited cells; 3) provide multiple slices from one animal for both control and experimental conditions and reduce the number of animals used. One major limitation of PCLS is that tissue preparation and section may introduce artifacts, but they can be monitored and controlled by allowing a stabilization period after slicing and by measuring lactate dehydrogenase [LDH] release and comparing untreated with treated slices. PCLS viability is excellent for 72 h and can be maintained for 7 days. We have established the doxycycline-inducible active TGFbeta transgenic mouse pulmonary fibrosis models. To test drug effect on TGFbeta-induced pulmonary fibrosis before they are used in the mouse model study, we prepared PCLS from the transgenic mice and cultured PCLS in vitro with/without doxycycline to induce pulmonary fibrosis in vitro. Our pilot study shows 3 day treatment with doxycycline induced pulmonary fibrosis as measured by a number of readouts and we are currently testing the effect of drugs to be used for in vivo studies. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This new method will be very useful for initial drug effect screening for a number of disease models before they are actually used in animal model studies. It will markedly reduce the number of animals used for in vivo studies. 
 
Description Collaboration on the epigenetic study of pulmonary artery hypertension in COPD 
Organisation Sichuan University
Department West China Medical School
Country China 
Sector Academic/University 
PI Contribution The MRC-supported grants have enabled us to collaborate with other researchers in the research area of epigenetic mechanisms of pulmonary diseases. We supported two visiting Chinese researchers from Sichuan University to conduct research in our laboratory on the epigenetic regulation of key genes in pulmonary artery hypertension in COPD, with one stayed for three months in 2014 and 3 months in 2015, respectively, and another stayed for 3 months in 2015 . They have generated some very useful pilot data. A Joint research project grant application to study the epigenetic mechanisms of cigarette smoke-induced imbalanced vasoactive gene expression in pulmonary hypertension was submitted to Medical Research Council in May 2015 but was rejected. A collaborative research project grant application to study molecular mechanisms of cigarette smoke- and hypoxia-induced imbalanced vasoactive gene expression in pulmonary hypertension has recently been submitted to the British Heart Foundation. This project involves the University of Cambridge as a partner and Sichuan University, University Paris XII, University of Barcelona and University of British Columbia as collaborators. Another joint grant application is being written by my collaborators at Sichuan University to be submitted to the Natural Science Foundation of China combining our knowledge and experience in epigenetic study and their knowledge and experience in animal model study. I have obtained funding for a research visit to Sichuan University to seek opportunities for further collaboration.
Collaborator Contribution My international partners in China are very experienced in the cellular, animal model and clinical studies of pulmonary artery hypertension in COPD. Two of them spent six months and three months with me to gain experience on epigenetic studies. The useful pilot data they have generated so far provide some of the basis for collaborative or joint grant applications with complimentary knowledge and research experience. My research partner at the University of Cambridge is internationally renowned for research in pulmonary hypertension and has made intellectual and technical contribution to the research work proposed in the project submitted to the British Heart Foundation and will be critically involved in the completion of the project if it is funded.
Impact Not yet.
Start Year 2014
 
Description Collaboration on the epigenetic study of pulmonary artery hypertension in COPD 
Organisation University of Cambridge
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution The MRC-supported grants have enabled us to collaborate with other researchers in the research area of epigenetic mechanisms of pulmonary diseases. We supported two visiting Chinese researchers from Sichuan University to conduct research in our laboratory on the epigenetic regulation of key genes in pulmonary artery hypertension in COPD, with one stayed for three months in 2014 and 3 months in 2015, respectively, and another stayed for 3 months in 2015 . They have generated some very useful pilot data. A Joint research project grant application to study the epigenetic mechanisms of cigarette smoke-induced imbalanced vasoactive gene expression in pulmonary hypertension was submitted to Medical Research Council in May 2015 but was rejected. A collaborative research project grant application to study molecular mechanisms of cigarette smoke- and hypoxia-induced imbalanced vasoactive gene expression in pulmonary hypertension has recently been submitted to the British Heart Foundation. This project involves the University of Cambridge as a partner and Sichuan University, University Paris XII, University of Barcelona and University of British Columbia as collaborators. Another joint grant application is being written by my collaborators at Sichuan University to be submitted to the Natural Science Foundation of China combining our knowledge and experience in epigenetic study and their knowledge and experience in animal model study. I have obtained funding for a research visit to Sichuan University to seek opportunities for further collaboration.
Collaborator Contribution My international partners in China are very experienced in the cellular, animal model and clinical studies of pulmonary artery hypertension in COPD. Two of them spent six months and three months with me to gain experience on epigenetic studies. The useful pilot data they have generated so far provide some of the basis for collaborative or joint grant applications with complimentary knowledge and research experience. My research partner at the University of Cambridge is internationally renowned for research in pulmonary hypertension and has made intellectual and technical contribution to the research work proposed in the project submitted to the British Heart Foundation and will be critically involved in the completion of the project if it is funded.
Impact Not yet.
Start Year 2014
 
Description Collaboration on the study of the antifibrotic effects of histone deacetylase inhibitors in an ex vivo lung slice model. 
Organisation University of Leicester
Department Department of Geography
Country United Kingdom 
Sector Academic/University 
PI Contribution Our group has developed an ex vivo lung fibrosis model with precision cut lung slices from transgenic mice and found it is useful for the assessment of the antifibrotic effect of histone deacetylase inhibitors. Professor Peter Bradding's group at the University of Leicester has also developed an ex vivo lung fibrosis model with human lung tissue slices. We are collaborating to investigate if the antifibrotic effect of histone deacetylase inhibitors observed in precision cut mouse lung slices can be observed in human lung tissue slices. The work is on going and good results from this collaborative study may accelerate clinical trials of the drugs.
Collaborator Contribution Professor Peter Bradding's group at the University of Leicester has also developed an ex vivo lung fibrosis model with human lung tissue slices.
Impact The work is on going.
Start Year 2016
 
Description Epigenetic study on radiation-induced lung fibroblast to myofibroblast differentiation in pulmonary fibrosis 
Organisation Third Military Medical University, China
Country China 
Sector Academic/University 
PI Contribution Our contributions to this collaboration is the knowledge and experience in epigenetic studies. An application has been made for a cost share scheme exchanges scheme between the Royal Society and the National Natural Science Foundation of China. If successful, the scheme will allow visits from both sides to the laboratories of the other sides in order to finalise details for collaborative research grant applications.
Collaborator Contribution Our collaborators have vast knowledge and experience in the research of radiation-induced lung fibroblast to myofibroblast differentiation in pulmonary fibrosis.
Impact Not yet.
Start Year 2014
 
Description Gordon Research Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact KEYNOT TALK AT THE GORDON RESEARCH SEMINAR AND CONFERENCE
Year(s) Of Engagement Activity 2017
 
Description Nottingham May Fest 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Questions and discussions from the public about the presentation and our research work as a whole.

Not yet.
Year(s) Of Engagement Activity 2014,2015