Endothelial cell biology and the initiation of inflammation

"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."

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.