Endothelial cell secretory granule exocytosis
Lead Research Organisation:
St George's, University of London
Department Name: UNLISTED
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).
People |
ORCID iD |
Tom Carter (Principal Investigator) |
Publications
Cookson EA
(2013)
Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis.
in Journal of cell science
Van Breevoort D
(2014)
STXBP1 promotes Weibel-Palade body exocytosis through its interaction with the Rab27A effector Slp4-a.
in Blood
Kiskin NI
(2014)
Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.
in PloS one
Conte IL
(2015)
Is there more than one way to unpack a Weibel-Palade body?
in Blood
Helassa N
(2015)
Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics.
in Scientific reports
Conte IL
(2016)
Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade body trafficking and exocytosis.
in Journal of cell science
Khan S
(2016)
Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.
in Biophysical journal
Schillemans M
(2018)
Weibel-Palade Body Localized Syntaxin-3 Modulates Von Willebrand Factor Secretion From Endothelial Cells.
in Arteriosclerosis, thrombosis, and vascular biology
Streetley J
(2019)
Stimulated release of intraluminal vesicles from Weibel-Palade bodies.
in Blood
Lenzi C
(2019)
Synaptotagmin 5 regulates Ca2+-dependent Weibel-Palade body exocytosis in human endothelial cells.
in Journal of cell science
Description | IMAGING GLUTAMATE IN THE BRAIN USING NOVEL FAST FLUORESCENT PROBES |
Amount | £615,802 (GBP) |
Funding ID | BB/S003894/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2018 |
End | 08/2022 |
Description | SGUL Medical Research Comittee fund |
Amount | £9,045 (GBP) |
Organisation | St George's University of London |
Sector | Academic/University |
Country | United Kingdom |
Start | 04/2016 |
End | 05/2017 |
Description | SGUL capital equipment fund |
Amount | £15,000 (GBP) |
Organisation | St George's University of London |
Sector | Academic/University |
Country | United Kingdom |
Start | 05/2014 |
End | 06/2015 |
Description | SGUL internal Postgraduate studentship |
Amount | £75,000 (GBP) |
Organisation | St George's University of London |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2013 |
End | 10/2018 |
Description | Bierings |
Organisation | Sanquin |
Country | Netherlands |
Sector | Private |
PI Contribution | Development at application of research tools to study the mechanism of secretory granule exocytosis |
Collaborator Contribution | Experimental work |
Impact | paper submitted for review |
Start Year | 2017 |
Description | Exocytosis in adult human heart endothelial cells |
Organisation | Imperial College London |
Department | National Heart & Lung Institute (NHLI) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We collaborate to study the mechanisms and regulation of WPB exocytosis in adult ECs of human origin |
Collaborator Contribution | We have gained access to sources of adult human heart ECs. The collaboration has led to us setting up new methods in the lab. |
Impact | PMID 23001355 PMID: 22898601 |
Start Year | 2009 |
Description | Software development for data aquisition |
Organisation | University of Strathclyde |
Department | Strathclyde Institute of Pharmacy & Biomedical Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We carry out testing of his programnmes and provide detailed feedback that allows Dr Dempster to improve his software. This has led to substantial improvements in the programme that have benefited the general scientific community who use this freeware software in their research. |
Collaborator Contribution | Dr John Dempster has written custom shareware software (Winfluor; http://spider.science.strath.ac.uk/PhysPharm/showPage.php?pageName=software_imaging) for high speed optical and electrophysiological data aquisition. He has provided a unique set of solutions for our needs, without his contribution we would not be able to do the work we do in the way we can now. |
Impact | Publications: PMID: 18252862 |
Start Year | 2013 |
Description | Ultrastructural analysis of Weibel-Palade bodies |
Organisation | Francis Crick Institute |
Department | Mill Hill Laboratory |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | Analysis of Weibel-Palade body composition and exocytosis by cell biological and fluorescence imaging approaches |
Collaborator Contribution | High resolution ultra structural analysis of Weibel-Palade bodies by cryo-EM |
Impact | Stimulated release of intraluminal vesicles from Weibel-Palade bodies. Streetley J, Fonseca AV, Turner J, Kiskin NI, Knipe L, Rosenthal PB, Carter T. Blood. 2019 Feb 13. pii: blood-2018-09-874552. doi: 10.1182/blood-2018-09-874552. [Epub ahead of print] PMID: 30760452 |
Start Year | 2013 |
Description | Zebrafish models of VWF secretion |
Organisation | St George's University of London |
Department | Population Health Research Institute |
Country | United Kingdom |
Sector | Hospitals |
PI Contribution | We provide expertise in cell biology |
Collaborator Contribution | Collaboratorbrings expertise in zebrafish gentics and husbandry |
Impact | none as yet |
Start Year | 2016 |
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 | 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 | Cell Physiology Workshop: providing advanced training in electrophysiology and Imaging techniques |
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 | 20 participants (postgraduates researchers to professorial level), 2 week virtual practical training course in advanced electrophysiology and Imaging techniques |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.digitimer.com/electrophysiology-imaging-a-virtual-plymouth-workshop-for-2021/ |
Description | Outreach Year 12 school children |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Took part in the SGUL Imaging resource Facility's out reach program called; Healthcare Dissected. This program is aimed at to introducing year 12 students to Healthcare and university research through a series of podcasts, and practical sessions in the imaging facility. |
Year(s) Of Engagement Activity | 2022 |