The effect of hypoxia and and reoxygenation on inflammation in human endothelial cells (360G-Wellcome-206974_Z_17_Z)

Ischaemia-reperfusion (I-R injury is a major cause of myocardial injury during acute coronary syndrome. Initial block of a coronary artery by plaque rupture and thrombosis leads to downstream myocardial ischaemia. Reperfusion is required to prevent extensive myocardial injury, but restoration of oxygen supply can trigger further injury to the ischaemic tissue. This represents a major unmet clinical problem. Inflammation is a component of I-R injury. Ischaemia and reperfusion trigger endothelial cell activation, followed by rapid recruitment and transmigration of neutrophils and further tissue damage. This project forms part of a wider effort to develop an in vitro model of vascular inflammation during I-R injury. I will characterise the response of human umbilical vein endothelial cells (HUVECs) to hypoxia followed by reoxygenation/normoxia, in the presence and absence of tumour necrosis factor (TNF)-alpha, to mimic an additional, tissue-derived inflammatory stimulus. HUVEC activation markers, including surface expression of P-selectin, E-selectin, ICAM-1 and VCAM, will be assessed using multi-colour flow cytometry and confocal microscopy. The consequence for neutrophil adhesion and transmigration will be assessed using confocal microscopy. These experiments will demonstrate the effect of hypoxia followed by normoxia of endothelial cell inflammatory responses, and increase our understanding of the mechanisms that underlie I-R injury.