Endothelial Cell Function and Weibel-Palade Bodies.

Lining all blood vessels are endothelial cells. Damage to these cells or changes in their behaviour can cause very important diseases, including Diabetes, Atherosclerosis, Coronary Artery Disease, Stroke, Deep Vein Thrombosis, Reperfusion Injury, and Pulmonary Embolism. Endothelial cells carry packets called Weibel-Palade bodies (WPB) filled with molecules that provide first aid to the endothelium, releasing their contents into the blood when the vasculature is injured. Excessive release can contribute to disease. We have been investigating the ways in which endothelial cells control how WPB and their content are made and released, and we aim to discover more about how this system works. Being able to change WPB will allow us to modulate their effect, and ultimately allow us to change endothelial behaviour. We hope that this will help to combat these diseases.

Overall Aim and Importance
Weibel-Palade Bodies (WPB) are the cigar-shaped multifunctional secretory granules of endothelial cells. They carry molecules necessary to haemostasis/thrombosis, inflammation, the control of vascular tonicity, and angiogenesis. They are thus of central importance in the endothelial contribution to vascular health and disease. Reduction of the level of their major component, Von Willebrands Factor, cause von Willebrands disease- the commonest inherited human bleeding disorder, but perhaps more importantly, higher levels of VWF are associated with Diabetes, Atherosclerosis, Coronary Artery Disease, Stroke, Deep Vein Thrombosis, Reperfusion Injury, and Pulmonary Embolism. The association of WPB components with this range of globally important diseases justifies significant investment in increasing our understanding of these organelles.

We have discovered a set of cellular components that control the formation and exocytosis and thus functioning, of these organelles, and we wish to understand how these controls work together to produce their final functional output. We wish to underpin and exploit a conceptual shift towards a new model where WPB formation and exocytosis are actively modulated.

Objectives
1. Determine how changes to WPB size caused by cellular controls produce different functional responses to different endothelial agonists.
2. Determine how different cellular controls act in concert to produce a final functional output from the WPB and thus control the activated endothelial cell phenotype.
3. Identify and characterise new controls on endothelial phenotype that operate through WPB.

Plan and methodology
Cellular controls so far discovered act to control WPB size; to control WPB maturation (and thus VWF multimerisation); to control WPB exocytosis. We will determine how these three aspects of control operating to modify the final functional output from these organelles combine to affect four key endothelial functions using in vitro assays.
Specifically, we will measure the effect of modulating machinery from more than one category on: WPB size and number; maturation and thus multimerisation of VWF; WPB exocytosis. The data from these will then determine which modulations will be tested for their effects on: Recruitment of plasma VWF to the activated cell surface; Recruitment of platelets by activated endothelial cells; Recruitment of leukocytes to the endothelial surface; Extravasation of leukocytes. These functional assays will be carried out in flow experiments, using both simple and more complex chambers.