Endothelial cell biology and the initiation of inflammation

Lead Research Organisation: University College London
Department Name: UNLISTED

Abstract

"One of the most visible signs of inflammation is the formation of pus. This is the accumulation of white blood cells at sites of injury, where they act to repair damage. To grab the white cells out of the mix of cells in rapidly flowing blood, the cells lining the blood vessels change so that instead of providing a smooth surface which promotes the flow of blood and prevents the formation of clots, they become selectively sticky. This selective stickiness comes from a special set of molecules that the lining endothelial cells express on their surface. The bound white cells accumulate on the surface of the endothelium, and then pass out between the endothelial cells into the tissue where they act. The endothelial cells ability to expose and then retrieve the sticky molecules, the selectins, in a regulated fashion, thus plays a big role in controlling the initiation of inflammation. The team of about half a dozen scientists at the CBU plus collaborators in the UK and abroad are trying to understand the molecular machinery that underpins this process. We study the problem in human cells taken from the lining of umbilical veins which we can grow in the laboratory. By understanding the machinery that controls this process, we hope to learn how to manipulate it so as to gain better control over inflammatory disease."

Technical Summary

At sites of inflammation, the surface of the endothelial cells lining blood vessels changes to recruit leukocytes. The regulated appearance of a series of molecules which interact with receptors on leukocytes is critical for the initiation of inflammation. The first membrane protein to appear is P-selectin. The disappearance of P-selectin is equally tightly controlled to avoid inappropriately prolonged inflammation. P-selectin is stored within the membrane of the Weibel-Palade body (WPB) within cells so that using regulated exocytosis of the WPBs, it can be placed on the plasma membrane within a few minutes. After its appearance at the cell surface, P-selectin is internalised and 50% then recycles back to WPBs for re-use, while 50% is targeted to the lysosomes for degradation. The functioning of P-selectin thus depends on a series of membrane trafficking events that control its itinerary. Further, the molecular machinery controlling its trafficking integrates P-selectin functioning with the inflammatory state of the cell by responding to physiologically generated signals. We are characterising the machinery controlling the itinerary of P-selectin within primary human endothelial cells obtained from the umbilical cord (HUVECS). We are determining the sequences within P-selectin that target this protein to the WPBs, that control its internalisation in endothelial cells, and that lead to its delivery to lysosomes or to its recycling. We are screening for proteins that interact with these targeting motifs by genetic as well as biochemical methods in addition to employing a candidate approach in identifying key players that control the behaviour of P-selectin. The response of this machinery to inflammatory signalling will be determined. Moreover, in addition to our analyses at the tissue culture level, we are collaborating with colleagues in Sheffield to analyse the effects of defects in the trafficking machinery on the ability of the endothelium to recruit leukocytes using intravital microscopy in mice. P-selectin storage depends on the formation of the WPB, which in turn depend on the haemostatic protein von Willebrands Factor (VWF). We are examining how VWF drives the formation of the WPBs, and in turn how that is affected in Von Willebrands disease- the commonest genetic haemostatic disorder, as well as in other inherited human bleeding disorders. These experiments use siRNA-mediated knock-down of disease genes followed by analyses in vitro using HUVECs of WPB formation and function. Our work on the WPB is revealing which defects in WPB formation might lead to haemostatic problems, and we collaborate with clinical colleagues at Hammersmith and Sheffield in the clinical interpretation of this data.

Publications

10 25 50

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Ferraro F (2020) Modulation of endothelial organelle size as an antithrombotic strategy. in Journal of thrombosis and haemostasis : JTH

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Ferraro Francesco (2015) ENDOTHELIAL CONTROL OF THE FUNCTIONAL ARCHITECTURE OF VWF in JOURNAL OF VASCULAR RESEARCH

Related Projects

Project Reference Relationship Related To Start End Award Value
MC_UU_12018/1 31/07/2013 30/03/2017 £1,079,000
MC_UU_12018/2 Transfer MC_UU_12018/1 31/07/2013 30/03/2017 £989,000
MC_UU_12018/3 Transfer MC_UU_12018/2 31/07/2013 30/03/2017 £925,000
MC_UU_12018/4 Transfer MC_UU_12018/3 31/07/2013 30/03/2017 £908,000
MC_UU_12018/5 Transfer MC_UU_12018/4 31/07/2013 30/03/2017 £1,560,000
MC_UU_12018/6 Transfer MC_UU_12018/5 31/07/2013 30/03/2017 £1,234,000
MC_UU_12018/7 Transfer MC_UU_12018/6 31/07/2013 30/03/2017 £1,070,000
 
Description A* STAR -LMCB collaboration
Amount £20,000 (GBP)
Organisation Agency for Science, Technology and Research (A*STAR) 
Sector Public
Country Singapore
Start 09/2013 
End 09/2015
 
Description Project grant
Amount £223,490 (GBP)
Funding ID PG/14/76/31087 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2015 
End 09/2018
 
Title Structured Illumination microscopy & platelets 
Description We developed methods for analysing platelets using super-resolution microscopy. This included sample preparation, bioinformatic image-analysis methods and data display. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact One published paper, one press release, and collaborations being set up to try to bring this diagnostic approach to patients. 
URL https://www.ncbi.nlm.nih.gov/pubmed/26806224
 
Description A* STAR Bioinformatics 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Bioinformatics institute (BII)
Country Singapore 
Sector Academic/University 
PI Contribution We are jointly funding and supervising a PhD student; I provide the cell biological expertise to this project
Collaborator Contribution They contribute half of the funding and computer-based Image analysis expertise to the project.
Impact This is a multi-disciplinary collaboratioh between cell biologists, clinicians, physicists, and bioinformaticians
Start Year 2013
 
Description Collaboration with Nightingale 
Organisation Queen Mary University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution High-throughput imaging, expertise in Weibel-Palade bodies
Collaborator Contribution manipulated endothelial cells
Impact Papers have been published resulting from this collaboration, and new data is being generated, for example by a new screen, that has not yet been used to generate outputs.
Start Year 2017
 
Description Mathematical Modelling of VWF and WPB 
Organisation University College London
Department Mathematics
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing biological data and intellectual input
Collaborator Contribution Mathematical expertise and bio-modelling expertise
Impact Manuscript Published: "Structural modelling hints...."2022.
Start Year 2015
 
Description platelet diagnostics 
Organisation National Physical Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell Biology and microscopy
Collaborator Contribution Royel Free: platelet expertise and access to patients NPL Expertise in super-resolution microscopy, image analysis software, and access to microscopes
Impact Grant applications have been made. A paper has been published: Westmoreland, D., Shaw, M., Grimes, W., Metcalf, DJ., Burden, JJ., Gomez, K., Knight, AE., Cutler, DF. (2016) Super-resolution microscopy as a potential approach to platelet granule disorder diagnosis. J Thomb. Haemost. Epub. This is multidisciplinary; Physics, Clinical Science, and Cell Biology
Start Year 2011
 
Description platelet diagnostics 
Organisation Royal Free London NHS Foundation Trust
Country United Kingdom 
Sector Public 
PI Contribution Cell Biology and microscopy
Collaborator Contribution Royel Free: platelet expertise and access to patients NPL Expertise in super-resolution microscopy, image analysis software, and access to microscopes
Impact Grant applications have been made. A paper has been published: Westmoreland, D., Shaw, M., Grimes, W., Metcalf, DJ., Burden, JJ., Gomez, K., Knight, AE., Cutler, DF. (2016) Super-resolution microscopy as a potential approach to platelet granule disorder diagnosis. J Thomb. Haemost. Epub. This is multidisciplinary; Physics, Clinical Science, and Cell Biology
Start Year 2011
 
Description super-resolution microscopy 
Organisation National Physical Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell Biological expertise
Collaborator Contribution Access to a pre-commercial microscope of improved resolution.
Impact Paper published in JTH., development of diagnostic methods. This is a multidisciplinary collaboration, involving physicists, clinicians, and cell biologists.
Start Year 2011
 
Description super-resolution microscopy 
Organisation Royal Free Hospital
Department Katharine Dormandy Haemophilia and Thrombosis Centre
Country United Kingdom 
Sector Hospitals 
PI Contribution Cell Biological expertise
Collaborator Contribution Access to a pre-commercial microscope of improved resolution.
Impact Paper published in JTH., development of diagnostic methods. This is a multidisciplinary collaboration, involving physicists, clinicians, and cell biologists.
Start Year 2011
 
Description Press Release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact On publication of the paper Westmoreland et al, UCL, NPL and MRC put out a press release describing the breakthrough. This was picked yup by a number of websites/online news sites etc, including: www.daijiworld.com/news; https://www.pinterest.com; www.microscopy-analysis.com; www.scoop.it/.; www.newsunited.com; www.sciencenewsline; Science Daily?; timesofindia.indiatimes.com; wn.com; sci24h.com; www.thehealthsite.com; www.icimagingsociety.org.uk; http://www.bloodline.net; https://flipboard.com/topic/bleeding; healthgoog.blogspot.com; https://www.latestnews360.com; http://www.britishsocietynanomedicine.org/news.html
www.hematology-sa.org (Saudi-Arabia)

In following up this discovery, I have spoken to the heads of the UK and USA Hermansky-Pudlak patients goups, I have given seminars and had discussions with clinical and basic science colleagues in the UK and elsewhere, and this work is now planned to be taken forward in new collaborations that are being set up in the UK and mainland Europe
Year(s) Of Engagement Activity 2016
URL https://www.ucl.ac.uk/news/news-articles/0116/040216-rare-bleeding-disorder
 
Description core staff briefing 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact the non-academic core staff of the LMCB attended a non-scientific laypersons version of my research, followed by a long Q&A.

an increase in morale via the increase in understanding of what their work is supporting.
Year(s) Of Engagement Activity 2009,2014