Endothelial cell secretory granule exocytosis

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

A thin continuous layer of cells covers the inside of all our blood vessels, separating blood from our tissues and cells. Unlike other cells in the body, blood does not clot when in contact with healthy endothelial cells. This unique and vital property stems from the secretion of molecules that prevent blood coagulation, and that rapidly dissolve blood clots if they do form. The endothelium is not however a one-trick-pony; in addition to controlling blood coagulation, they play essential roles in regulating blood flow, tissue repair and growth, and inflammation within the vasculature and adjacent tissues. Endothelial cells control all of these different processes through the secretion of a large number of different molecules. In effect, the vascular endothelium functions as a highly specialised multi-tasking secretory machine that responds rapidly to changes in its local environment. Alterations to the normal secretory function of endothelial cells are though to contribute to an increased risk of hypertension and atherosclerosis; major causes of stroke and heart attacks. Our research is aimed at trying to understanding the cellular mechanisms that regulate secretion from these cells. We focus particularly on the secretion of peptides and proteins involved in the control of blood coagulation (von Willebrand factor and tissue plasminogen activator) and inflammation (p-selectin and small cytokines such as interleukin-8). We combine biochemical, molecular, cell biological and biophysical approaches to directly analyse the synthesis, storage and secretion of these molecules in single endothelial cells. Understanding how the secretory function of endothelial cells is controlled under normal conditions will shed light on the changes that occur during disease and help us to develop new strategies for the treatment of vascular disorders.

Technical Summary

Vascular endothelial cells play a crucial role in the regulation of blood flow, blood clotting and inflammation through the secretion of a wide range of vasoactive, pro- or anti-coagulant and inflammatory molecules. Many of these molecules are of great clinical importance including the anti-coagulant tissue plasmonogen activator (tPA) and the pro-coagulant and inflammatory molecules von Willebrand factor (vWF) and p-selectin. In addition to their physiological roles, vWf and p-selectin have been implicated in the development of vascular disease (atherosclerosis). Our research is aimed at understanding the mechanisms and regulation of secretion of vWF, p-selectin and tPA from human endothelial cell. These studies will lead to a better understanding of the contribution of endothelial cells to the regulation of blood flow, blood clotting and intravascular inflammation.||Our specific aims are (1) to provide a detailed kinetic description of hormone and calcium evoked exocytosis of two distinct classes of secretory organelle found within endothelial cells, the Weibel Palade body (WPb) that contains vWF and p-selectin, and the tPA containing organelle, (2) to determine the Ca2+-dependence of tPA and WPb secretory organelle exocytosis, (3) to examine the contribution of cytoskeletal elements (microtubules; MTs, and actin) to granule movement and recruitment during stimulation, (4) to determine the distribution of secretory organelle release sites and the fate of secreted proteins and (5) to identify the SNARE proteins involved in WPb exocytosis.||We use cultured human umbilical vein endothelial cells (HUVEC) as a model system in which to study endothelial secretion. Our approaches to investigating endothelial exocytosis include electrophysiological measurements of endothelial cell membrane surface area, direct optical (epi-fluorescence, total internal reflection fluorescence or confocal) recordings of fluorescent WPb and tPA organelles in living HUVEC, and biochemical assays of vWF, proregion and tPA release. Our studies take advantage of morphological peculiarities of WPb and of endothelial cells (EC) in general. (1) The large size of WPb allows individual WPb to be detected as fast discrete steps in membrane capacitance (Cm) in high-resolution patch clamp capacitance recordings. (2) Their unusual shape (rod like) and large size make WPb easy to identify using optical (bright field or fluorescence microscopy) approaches and for cell biological investigations. (3) Our ability to label WPb with fluorescent proteins enables the formation, movement and exocytosis of individual WPb to be analysed in detail. The relatively limited numbers of WPb per cell (up to a few hundred), allow the behaviour of each individual WPb within a population to be investigated within single cells. (4) With the exception of the nuclear region, HUVEC (and EC in general) are very flat and thin (<1um), making these cells ideal for optical studies of fluorescent organelles. Together with the physiological and pathological importance of the secreted factors, the ease of culture and amenability to transfection of HUVEC (using nucleofection procedures), these endothelial cells provide an excellent model system in which to study secretory granule biogenesis, trafficking and secretion.||Our research program draws upon collaborations with groups within NIMR (Dr Matthew Hannah (Molecular Neuroendocrinology), Dr David Ogden (Neurophysiology), Drs Justin Molloy, Gregory Mashanov, Mike Anson, John Corrie and Gordon Reid (Physical Biochemistry)) as well as other UK (Dr Paul Skehel (Edinburgh University), Dr C.P.D. Wheeler-Jones (Royal Veterinary College, London), Dr V O Connor (Southampton University), and overseas institutions Dr G. Zupancic (University of Ljubljana, Slovenia).

Publications

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Berriman JA (2009) Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells. in Proceedings of the National Academy of Sciences of the United States of America

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Cookson E (2012) Properties of the Weibel-Palade Body Fusion Pore in Biophysical Journal

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Gregory Mashanov (2007) Single fluorophore tracking of P-selectin-EGFP during exocytosis of Weibel-Palade bodies. in European Biophysics Journal

 
Description Endothelial cell secretory granule exocytosis
Amount £974,719 (GBP)
Funding ID MC_PC_13053 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 03/2018
 
Title LAMP-1-dt-Tom 
Description Lysosoaml marker prtoein. Expression allows direct visualisation of lysosomes in living cells 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact publication: PMID: 18252862 
URL http://europepmc.org/abstract/MED/18252862
 
Title MyRIP 
Description Fusion of WT full length MyRIP with EGFP 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Use of reagent resulted in greater understanding of the mechanisms regulating Von Willebrand factor secretion from endothelial cells 
 
Title P-selectin construct 
Description Leukocyte adesion molecule. Expression labels secretory granules in endothelial cells and other cell types. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Publication: PMID: 18252862 
URL http://europepmc.org/abstract/MED/18252862
 
Title Slp4-a 
Description fusion protein of full length WT Slp4-a with EGFP, mCherry 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Use of reagent led to a greater understanding of the mechansims regualting von Willebrand secretion from endothelial cells 
 
Title VWF constructs 
Description VWF-propolypeptide fused to different fluorescent proteins (EGFP, YFP, CFP, mRFP, mCherry, tdTomato) 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Has allowed direct visualisation of secretory granules in living endothelial cells in conjunction of other markers of the secretory pathway. 
 
Title eotaxin-3 m EGFP 
Description eotaxin-3 fused to mEGFP 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Has allowed the direct visualisation of the behaviour of this small cytokine during secrerory granule formation and release during exocytosis in endothelial cells 
 
Title interlukin-8 mCherry 
Description interlukin-8 fused to mCherry 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Has allowed the direct visualisation of this cytokine within the secretory pathway of endothelial cells. Revealed novel mde of endothelial secretion. 
 
Title tPA-EGFP 
Description Sectreted protein. Expression labels two distinct granule populations in endothelial cells. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact none during current review period 
 
Description Analysis of secretory kinetics 
Organisation University of Paris - Descartes
Country France 
Sector Academic/University 
PI Contribution My group carries out the basic research and builds and developes the experimental setups used, with advice from our collaborator, Dr David Ogden (formerly in Neurophysiology at NIMR.)
Collaborator Contribution This collaboration has provided significant technical and intellectual advice on the development of experimental techniques and the interpretation of data.
Impact Publications; PMID: 15831502, PMID: 17540703
 
Description Biochemical analysis of exocytosis in endothelium 
Organisation University of Southampton
Department Faculty of Natural and Environmental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide the experimental model system, reagents and data analysis facilities.
Collaborator Contribution Practical experimental and intellectual contributions
Impact Publication of meeting Abstracts (Biochemical Soc Transations)
 
Description Developement of reagents for research 
Organisation University of Edinburgh
Department Centre for Neuroregeneration (CNR)
Country United Kingdom 
Sector Academic/University 
PI Contribution We carry out experimental research with these reagents and have used them as a base for developing further reagents with our lab (fluorescent colour swops and mutants).
Collaborator Contribution Dr Paul Skehel has developed reagents (tPA-EGFP, P-selectin-DsRed) that have been extensively used in our research. He is also involved in the development of animal models for studying secretion in vivo.
Impact Publications: PMID: 18252862
 
Description Statistical analysis of secretory kinetics 
Organisation University of Ljubljana
Country Slovenia 
Sector Academic/University 
PI Contribution My group provides the experimental data for which the statistical analysis programs are written. The programmes are written by DR Gregor Zupancic, University of Ljubljana. Feedback and beta testing of programmes are carried out in the lab.
Collaborator Contribution This collaboration has led to the development of software programs essential for the the quantitative analysis of the kinetics and mechanisms underying secretory granule exocytosis
Impact Publications PMID: 17540703
 
Description TIRF imaging of exocytosis in endothelial cells 
Organisation Yale University
Department Department of Cell Biology
Country United States 
Sector Academic/University 
PI Contribution We are making custom fluorescence constructs for high resolution TIR imaging of exocytosis of WPBs
Collaborator Contribution We have gained reagents and will in the futre gain joint publications
Impact No out comes as yet, but in the future this will result in joint opublications.
Start Year 2013
 
Description Torok 
Organisation St George's Hospital
Department Institute of Biomedical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution test genetically encoded ca indicators
Collaborator Contribution Make genetically encoded ca indicators
Impact none
Start Year 2013
 
Description Ultrastructural studies of Weibel Palade bodies 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution Experimental data, Intellectual input
Collaborator Contribution Publications, intellectual input
Impact Publications PMID: 19805028
 
Description WPB formation and secretion 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution reagents, experimental research, intellectual
Collaborator Contribution Reagents, intellectual input, practical expeimental aid
Impact Publications PMID: 15831502 PMID: 17540703 PMID: 18252862 PMID: 19258324 PMID: 19805028
 
Description Advanced techniques workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Practical teaching of advanced electrophysiological and imaging techniques to postgraduate, postdoctoral researchers and academics. Course provided excellent forum for exchange of ideas and knowledge between experienced and junior researchers.
Year(s) Of Engagement Activity 2017
URL http://www.mba.ac.uk/microelectrode-techniques-cell-physiology
 
Description Advanced techniques workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Teach groups of 3-5 postgraduate students advanced imaging and electrophysiology techniques

Students often return as teachers and demonsrtators on the course in later years.

Students forge collaborative links with other students and teachers to further their careers.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
URL http://www.mba.ac.uk/microelectrode-techniques-for-cell-physiology/
 
Description Advanced techniques workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact training next generation of researchers
Year(s) Of Engagement Activity 2016
URL http://www.mba.ac.uk/microelectrode-techniques-cell-physiology
 
Description School Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Lecture on blood clotting Local schools 30 minutes feedback positive

Non other than to elicit questions and interested from the audiance.
Year(s) Of Engagement Activity 2006,2010
 
Description University of the 3rd Age 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Lecture on Blood clotting Audience: University of the 3rd Age 30 minutes positve feedback

Non other than to elicit questions and interested from the audiance.
Year(s) Of Engagement Activity 2006