Endothelial dysfunction is an early stage in atherosclerosis and is associated with oxidative stress, which occurs when reactive oxygen species are not fully compensated by cellular antioxidants. This study investigated the effects of hydrogen peroxide in a human endothelial cell model. EA.hy926 cells incubated for 1 hour at 37°C with increasing concentrations of hydrogen peroxide were investigated for DNA damage ('Comet' assay), changes in mitochondrial activity (MTT assay), apoptosis and necrosis (Annexin V assay), cell growth and senescence (changes in morphology, cell size, pH6 specific B-galactosidase (S-Gal) and telomere shortening). Changes in the expression of mRNA transcripts of thrombotic (tissue factor); fibrinolytic (tissue phasminogen activator and plasminogen activator inhibitor-1) and adhesion (ICAM-1) proteins were investigated using RT-PCR. Low (≤50muM) concentrations of hydrogen peroxide induced detectable, but reversible, DNA damage; which was accompanied only by a small decrease in mitochondrial activity. Intermediate levels of oxidative stress (<200muM H2O2) resulted in a dose-dependent effect on DNA damage and mitochondrial function. Exposure to hydrogen peroxide concentrations equal to or above 200muM caused immediate changes in a sub-set of the cells, including apoptosis (n=4). In the surviving cells proliferative ability was reduced, and senescent-like cells developed; indicated by an increase in S-Gal positive cells, a skewed increase in cell size and the appearance of 'giant' cells, but not by changes in telomere length. Tissue Factor and ICAM-1 mRNA levels increased with a corresponding increase in surface ICAM-1 protein expression, but there was no effect on either of the fibrinolytic factor transcripts (n=3). This study demonstrated that endothelial cells surviving an oxidative insult showed an altered phenotype becoming more adhesive and possibly more thrombogenic in parallel with the appearance of senescence. In vivo, these changes would accelerate the progression of atherosclerothrombosis and lead to an impaired ability to repair further injury to the endothelium.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:403515 |
| Date | January 2003 |
| Creators | Tull, Samantha Petula |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/29877 |
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