Endothelial cell secretory granule exocytosis

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

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).