Molecular mechanisms of reduced expression of the angiostatic chemokine IP-10 in idiopathic pulmonary fibrosis

Lead Research Organisation: University of Nottingham
Department Name: Division of Respiratory Medicine

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disorder characterised by irreversible scarring of the lung and has no effective therapy. Excessive fibroblast (a cell type) growth and scar formation are the most important features of the disease and require new blood vessel formation to accommodate the nutritional need. In normal lung the new blood vessel formation is inhibited by an inhibitor protein called IP-10. However, in IPF lung, IP-10 production is reduced, leading to new blood vessel formation and unopposed fibroblast growth and scar formation. Why IP-10 expression is faulty in IPF lung is unclear and is an important area to study. We believe that key processes in IP-10 expression are defective in IPF. In the studies proposed here we will compare fibroblasts from IPF patients with normal cells to examine the molecular mechanisms regulating the IP-10 expression and identify the defective processes in IPF. This will help us to understand the disease better and may lead to the development of approaches to restore IP-10 expression as a new treatment for IPF.

Technical Summary

Idiopathic pulmonary fibrosis (IPF) is a severe, progressive and lethal fibrotic lung disorder with less than a 50% five-year survival rate. IPF is characterised by dysregulated repair of inflammatory injury, fibroproliferation, and excessive collagen deposition. The etiology of IPF is unknown and the main therapy with anti-inflammatory corticosteroids is ineffective. Thus novel therapeutic strategies to modulate specific mechanistic pathways that lead to fibrosis are needed.
Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is critical in both physiological and pathological processes. Fibroblast proliferation and excessive collagen deposition are the most important pathological hallmarks of IPF and increasing evidence suggests that angiogenesis is a critical event that supports these processes. Angiogenesis is regulated by an opposing balance between angiogenic and angiostatic factors. IL-8 and interferon (IFN) ?-inducible protein of 10 kD (IP-10) of the CXC chemokine subfamily possess potent angiogenic and angiostatic properties, respectively. Recently, an imbalance in the levels of angiogenic (i.e. IL-8) and angiostatic (i.e. IP-10) chemokines that favours net angiogenesis has been demonstrated in both animal models and tissue specimens from patients with IPF and a key role of IP-10 in regulating angiogenesis in IPF has been suggested. In our preliminary studies using fibroblasts from IPF patients (F-IPF) and control subjects with nonfibrotic lung (F-NL) we have shown that compared to F-NL, cytokine-induced IP-10 protein production, mRNA expression and mRNA stability are reduced in F-IPF, but the molecular mechanisms remain to be explored.
We hypothesise that key processes in IP-10 gene transcriptional and posttranscriptional regulation are defective in IPF. We will use primary cultures of F-IPF and F-NL and compare their responses to IFN? and TNFa to test the hypothesis and identify the sites(s) of the defect(s). In the transcriptional studies, we will explore the expression, activation and IP-10 promoter binding of transcription factors and the expression, activity and recruitment to the IP-10 promoter of histone acetyltransferases and histone deacetylases. In the posttranscriptional mechanism studies, we will explore the expression, activation and RNA binding of AU-rich element (ARE) binding proteins and the involvement of the intracellular kinase pathways.
This application builds on our expertise in molecular studies of gene expression and will answer fundamental questions regarding IPF pathogenesis. The understanding of the molecular mechanisms of reduced IP-10 expression may identify specific therapeutic targets and lead to the development of molecular approaches to restore IP-10 expression as a novel therapy for IPF.
 
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 Myofibroblast differentiation in idiopathic pulmonary fibrosis: epigenetic mechanisms and therapeutic targeting by cAMP
Amount £563,754 (GBP)
Funding ID MR/K003259/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 02/2013 
End 07/2016
 
Description Visiting Fellowship
Amount £6,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 05/2014 
End 08/2014
 
Description Wellcome Trust Project Grant (Role of aberrant histone and DNA methylation in the downregulation of cyxlooxygenase-2 and IP-10 in idiopathic pulmonary fibrosis)
Amount £220,540 (GBP)
Funding ID 088751 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 09/2012
 
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 Dr Xu Mingyan 
Organisation Xiamen University
Department Medical College Xiamen
Country China 
Sector Academic/University 
PI Contribution Idea development, supply of materials, joint funding and joint publication
Collaborator Contribution Idea development, joint funding and joint publication
Impact A paper has been published (PMID: 23583295), which provide further evidence that lung fibroblasts from IPH patients are epigenetically deregulated.
Start Year 2011
 
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 mechanisms of pulmonary fibrosis 
Organisation University of Pittsburgh
Department Division of Pulmonary, Allergy, and Critical Care Medicine
Country United States 
Sector Academic/University 
PI Contribution We conducted cellular and molecular studies using the cells to explore the difference in the expression of key anti-fibrotic genes between normal and diseased cells and to dissect the epigenetic mechanisms for the repression of the genes in pulmonary fibrosis.
Collaborator Contribution Providing fibroblasts from the lungs of patients with idiopathic pulmonary fibrosis and from non-fibrotic lungs.
Impact Four publications have resulted from this collaboration.
Start Year 2006
 
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