Oke, Adedayo Olufunlola; (2002) The effects of antioxidant vitamins on the expression of tissue factor and tissue factor pathway inhibitor genes in human monocytic and endothelial cell lines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Oxidative stress has been implicated in the pathogenesis of various diseases including cancer, septic shock, stroke, hypertension and atherosclerosis. This manifests itself in oxidative modification of low-density lipoprotein (LDL) and the expression of specific proteins such as tissue factor (TF). It has been claimed that some of these effects can be reversed by dietary antioxidants and that at least some of these actions may be at gene level in vascular cells. The aim of the project was to study the effects of minimally oxidised low-density lipoprotein (mmLDL) and the bacterial endotoxin - lipopolysaccharide (LPS) on the expression of genes for TF and TF pathway inhibitor (TFPI) in a human monocytic cell line (THP-1) and the cell line ECV 304 (once thought to be of endothelial origin). The effect of antioxidant vitamins on the expression of these genes was elucidated. The action of the antioxidant vitamins on TF protein activity was also investigated. The principal antioxidants studied were ascorbate, α-tocopherol and lutein. The mode of action of the antioxidants was also studied through the involvement of selected nuclear transcription factors (NFkB, CREB, ELK-1 and HIF-1). In this study, Quantitative Competitive RT-PCR (QC RT-PCR) was successfully developed and applied for the determination of the changes in TF and TFPI expression. Some of the results were confirmed using Northern blot techniques. There was simultaneous expression of TF and TFPI mRNA in both THP-1 and ECV 304 cells after stimulation with a range of effectors. When an increase in TF expression was evident, a corresponding decrease in TFPI expression was noted in the case of LPS stimulation. The study confirmed that mmLDL and LPS are potent inducers of TF expression in THP-1 and ECV 304 cells. The treatment of cells with antioxidant vitamins significantly suppressed the induction of the TF gene and also induced the expression of TFPI mRNA. LPS and mmLDL also increased TF pro-coagulant activity on the surface of the cells and this activity was suppressed by antioxidants. Also, the antioxidants have effects on some of the nuclear transcription factors studied. The timing of the effects of antioxidants was given particular attention.

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