Endothelial cell biology and the initiation of inflammation

Lead Research Organisation: MRC Cell Biology Unit

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
 
Description Development Gap Fund
Amount £80,000 (GBP)
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 09/2012 
End 10/2013
 
Description MRC Development Gap funding
Amount £40,000 (GBP)
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 10/2010 
End 04/2011
 
Description In Vivo functionality 
Organisation University of Sheffield
Department Department of Cardiovascular Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Endothelial cell biology in vitro
Collaborator Contribution Endothelial function in vivo
Impact Doyle et al, Blood; 2011 Which led to Translational work via MRCT, leading to two grants from MRCT and to patent applications etc.
Start Year 2010
 
Description VWF and Angiogenesis 
Organisation Imperial College London
Department British Heart Foundation Centre of Research Excellence
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Cell Biological expertise
Collaborator Contribution Expertise and unique reagents plus access to patients
Impact Papers published: 1. Starke RD, Ferraro F, Paschalaki KE, Dryden NH, McKinnon TA, Sutton RE, Payne EM, Haskard DO, Hughes AD, Cutler DF, Laffan MA, Randi AM (2011) Endothelial von Willebrand factor regulates angiogenesis. Blood 117: 1071-1080 2. Starke RD, Paschalaki KE, Dyer CE, Harrison-Lavoie KJ, Cutler JA, McKinnon TA, Millar CM, Cutler DF, Laffan MA, Randi AM. (2013) Cellular and Molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells. Blood. 121: 2773-84 Other publications 10.1136/heartjnl-2011-300920b.39 (2011) BLOOD-DERIVED ENDOTHELIAL PROGENITOR CELLS FROM VON WILLEBRAND\'S DISEASE PATIENTS DEMONSTRATE THAT VON WILLEBRAND FACTOR REGULATES ANGIOGENESIS 000331833601394 (2013) Defective angiopoietin-2 release from von Willebrand disease patients\' blood outgrowth endothelial cells
Start Year 2007
 
Description modulating endothelial function 
Organisation Syntaxin
Country United Kingdom 
Sector Private 
PI Contribution expertise
Collaborator Contribution Unique reagents
Impact We have achieved a significant change in endothelial function, using novel proprietary pharmaceuticals. The company paid for their analysis in our system.
Start Year 2008
 
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
 
Title Methods of inhibiting inflammation 
Description identification of CD63 as a potential target for modulation of the inflamatory response of endothelial cells. 
IP Reference Insufficient Information 
Protection Patent granted
Year Protection Granted 2012
Licensed No
Impact DGF funding to my lab.
 
Description Discussion- Syntaxin, UK 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact hosted discussion with UK biotech company

funding of preliminary experimental work in my lab.
Year(s) Of Engagement Activity 2008,2009
 
Description Speaker at MRC -Showcase 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Showcasing our work to UK industry

Unclear, possibly led to interaction with Syntaxin.
Year(s) Of Engagement Activity 2007
 
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