Thrombin & its Major Receptor in Transforming Growth Factor-beta Release and Adhesion Formation in Pleural Infection

Lead Research Organisation: University College London
Department Name: Medicine

Abstract

Pleural infection (commonly known as pleurisy) affects over 65,000 patients each year in the UK and USA, with a high mortality of 20%. Pleurisy is characterized by pus collection in the chest. Drainage of the pus is crucial to successful management, but is often difficult as pus is often locked into multiple pockets by fibrous septations. Understanding the pathophysiology of formation of these septations will allow strategies to target their formation. Our recent experiments suggest that pleurisy induces accumulation of thrombin (an enzyme involved in blood clotting) in the chest, which stimulates transforming growth factor (TGF)-beta release from mesothelial cells (that line the pleural cavity) in vitro. This is important as we have previously showed that TGF-beta can potently stimulate adhesions and fibrosis.

The proposed project will bring together the expertise of five established researchers on TGF-beta and mesothelial cell biology (Dr Lee), PARs (Drs Chambers & Moffatt), pleural disease (Dr Davies) and pulmonary infection (Dr Brown). The proposed studies will build on our preliminary data and test the hypothesis that thrombin-induced release of TGF-beta plays an important role in the formation of adhesions / septations in pleurisy, using a variety of in vitro techniques and in vivo experimental models.

Technical Summary

Pleural infection kills over 10,000 patients in the UK and USA annually. Pleural infection is characterized by fibrous adhesions/septations which prohibit evacuation of pus - resulting in ongoing sepsis. No effective medical therapy exist to reduce adhesion formation.

We have shown that intrapleural administration of transforming growth factor (TGF)-beta potently induced collagen deposition and development of pleural adhesions. Active TGF-beta levels are significantly elevated in pleural fluid during pleural infection. What stimulates TGF-beta release during pleural infection is unknown.

Extravasation and activation of coagulation factors is common following pleural injury. Our preliminary data suggest that intrapleural infection induces thrombin accumulation, and the major thrombin receptor, proteinase-activated receptor (PAR)-1, is expressed on pleural mesothelial cells. In vitro, thrombin stimulates TGF-beta release from cultured mesothelial cells.

We hypothesize that pleural infection induces intrapleural accumulation of thrombin, which stimulates TGF-beta release from mesothelial cells, with resultant procollagen production and adhesion formation. The proposed study aims to show (i) that pleural infection leads to intrapleural accumulation of thrombin and TGF-beta; (ii) that thrombin induces mesothelial cell TGF-beta production via PAR1; and (iii) the effect of thrombin-induced TGF-beta on procollagen production and adhesion formation during pleural infection.

Animal studies: (i) To define the effects of pleural infection on pleural accumulation of thrombin, TGF-beta and collagen, C57BL/6 mice will be inoculated intrapleurally with S. pneumoniae, S. aureus or saline. Thrombin, TGF-beta and procollagen peptide levels will be measured in the pleural lavage; and pleural adhesion assessed macroscopically and histologically. (ii) Wild type and PAR1-/- mice inoculated with bacteria will be treated with a thrombin inhibitor, a PAR1 antagonist, or an anti-TGF-beta antibody to confirm the ?necessity? of thrombin, PAR1 and TGF-beta in adhesion formation. In vitro studies: (i) To confirm that thrombin induces TGF-beta release via PAR1, mesothelial cells will be cultured with thrombin, with or without antagonists to PAR1 or its downstream mediators. Human studies: (i) To confirm the relevance of the experimental findings in humans, thrombin and TGF-beta levels will be measured in pleural fluid from patients with pleural infection, and correlated with disease severity. (ii) Human infective pleural fluid will be added to cultured mesothelial cells, with or without a thrombin inhibitor, PAR1 antagonist and anti-TGF-beta antibody. Procollagen synthesis and gene expression will be assessed.

This study will provide insight on the pathophysiology of pleural adhesion formation and may allow the design of strategies to inhibit adhesion formation to improve patient care.

Publications

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Wilkosz S (2012) Characterization of a new mouse model of empyema and the mechanisms of pleural invasion by Streptococcus pneumoniae. in American journal of respiratory cell and molecular biology

 
Description Clinical Reference Group membership
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact responsible for the design and implementation of national commissioning for subspecialty care for specific areas of respiratory medicine- implemented subspecialty care for three separate conditions across England
 
Description Lecture to GulfThoracic conference
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact I gave two lectures on the care of pneumonia patients at the GulfThoracic conference, Dubai; by educating the healthcare workforce these should improve provision of care for these patients in the Middle East
 
Description NICE pneumonia guidelines
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description Royal College Regional training day lectures (three in total, a fourth in Oct 2014)
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact I have given 2012-2013 three lectures to Royal College of Physician regional training days on various aspects of clinical care for patients with pneumonia; a fourth lecture is planned for the national training conference October 2014
 
Description Clinical PhD fellowship
Amount £228,751 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 10/2015
 
Description Project grant
Amount $50,000 (AUD)
Organisation Westcare Foundation 
Sector Charity/Non Profit
Country United States
Start 09/2013 
End 03/2014
 
Title mouse model of post-pneumonic empyema 
Description we have developed a model of Streptococcus pneumoniae pneumonia that consistently results in empyema, allowing further research into the mechanisms involved. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2012 
Provided To Others? Yes  
Impact Published in 2012 - 34. Wilkosz, S., Edwards, L. A., Bielsa, S., Hyams, C., Taylor, A., Davies, R. J. O., . . . Lee, Y. C. G. (2012). Characterization of a New Mouse Model of Empyema and the Mechanisms of Pleural Invasion by Streptococcus pneumoniae. AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, 46(2), 180-187. doi:10.1165/rcmb.2011-0182OC 
 
Description Pathogenesis of empyema 
Organisation University of Perth
Department Faculty of Medicine
Country Australia 
Sector Academic/University 
PI Contribution Collaboration on the design of experiments, research questions and providing bacterial strains
Collaborator Contribution Obtaining funding from Australian sources Performing the majority of the experimental work
Impact Small grant: Westcare project grant Translational research in pleural infection. Prof G.Lee, J.S.Brown. A$50000. Paper: J.F.Varano della Vergiliana, S.M.Lansley, J.M.Porcel, S.Bielsa, J.S.Brown, J.Creaney, S.Temple, G.W.Waterer, Y.C.G.Lee. 'Bacterial infection elicits heat shock protein 72 release from pleural mesothelial cells.' In press PLoS One 2013.
Start Year 2011