Sacre, Sandra Michelle; (2000) Endothelial cell annexins and oxidative stress. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Annexins are a family of calcium-binding proteins that bind membranes containing negatively charged phospholipids in a calcium-dependent manner. To date 12 human annexins have been identified, many of which are expressed in vascular endothelial cells. Annexins 1 and 5 have been suggested to play a role in the inhibition of endothelial cell cPLA₂, but this may be by substrate sequestration rather than a direct interaction between the proteins. Annexin 6 has been shown in vitro to produce a less thrombogenic surface when bound to phospholipids on the cell surface of endothelial cell membranes. The most interesting suggestion is that annexin 2 is a receptor for plasminogen and tissue plasminogen activator on the cell surface of human umbilical vascular endothelial cells (HUVECs), acting as a catalyst for the conversion of plasminogen to plasmin. When the vascular endothelium is damaged by oxidative stress this can lead to diseases such as atherosclerosis and hypertension. This damage is accompanied by changes in calcium responses and transcriptional regulation. This thesis reports an investigation into how the behaviour and expression of certain annexins is changed as a consequence of oxidative injury to vascular endothelial cells. HUVECs were cultured in 40% oxygen in a custom-built chamber to induce chronic oxidative stress over a period of 12 days. Immunostaining of annexins revealed altered patterns of translocation following stimulation with a range of different agonists, in cells cultured under normal and hyperoxic conditions. To investigate the underlying causes of these phenomena, changes in cellular levels of calcium, protein phosphorylation, MAP kinase activation and tyrosine phoshorylation were examined in oxidatively stressed cells. Levels of annexin mRNA and protein expression were also examined in HUVECs. The major findings of this work showed that annexin protein expression was unchanged, although prehaps with modest changes in mRNA expression for some annexins, in oxidatively stressed HUVECs. Changes in calcium mobilisation and other signalling events induced by oxidative stress were observed in conjunction with changes in annexin localisation. Annexin 5 showed the most dramatic change by translocating to the nuclei of oxidatively stressed cells, when it would not normally be seen in the nucleus. Studies in this thesis together with published work, indicate protein phosphorylation as being a major event leading to the changes observed in calcium signalling and annexin localisation in oxidative stressed HUVECs.

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