The effects of oxidative stress on vascular smooth muscle cells
thesisposted on 15.12.2014, 10:31 by Elizabeth Mary. Woolaghan
The effect of oxidative stress on vascular smooth muscle cell (VSMC) function was investigated since these cells are an important participant in atherogenesis, and previous data regarding the effects of oxidative stress on VSMC function are inconclusive or contradictory.;Oxidative stress has been shown to cause both DNA damage and to stimulate cell growth in various cell types, although work in VSMC has been limited. Therefore, the effects of H2O2 on VSMC proliferation were studied using H2O2 as a well defined source of ROS. H2O2 inhibited serum-stimulated 3H-thymidine uptake in a dose dependent manner in SMC, by preventing cells from entering S-phase of the cell-cycle.;NFB appears to play a central role in mediating the cellular effects of oxidative stress, hence the effects of oxidative stress on NFB regulation and the role IB plays in mediating these effects in human VSMC were investigated. Using immunofluorescent microscopy, active NFB was detected in the nucleus of both H2O2 and TNF- treated cells. Whilst activation of NFB by TNF- involved the classical pathway of IB degradation, this degradation was not observed in H2O2 treated cells. This suggests that H2O2 utilises an alternative pathway of NFB activation that does not involve IB degradation.;Finally, the effects of H2O2 on gene expression in SMC were studied using a PCR-based differential display method. A number of differentially expressed genes were identified using this technique following H2O2 treatment. However, subsequent analysis by Northern blotting demonstrated that many of these genes were not differentially expressed. In light of these results,, efforts were focused on determining the reasons for such a high level of false positives. A number of modifications to the technique were developed to reduce the generation of non-specific PCR products, reduce the presence of co-migrating cDNA species and improve the efficiency of identifying and screening truly differentially expressed genes associated with oxidative stress.