Skene, C; (2014) The role of nitrosative and metabolic stress in vascular cell senescence. Doctoral thesis , UCL (University College London).

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Abstract

Vascular cell senescence has been observed in vascular healing, atheroma and advanced age. Other groups have demonstrated that senescence is a process characterized by metabolic dysfunction. We therefore investigate whether it may occur as a consequence of metabolic stress. Controversy existed as to whether nitric oxide was able to prevent senescence in vascular cells. As nitric oxide is able to inhibit mitochondrial respiration, we investigated its effects both as an oxidant and metabolic stressor. We observed that cells exposed to nitric oxide donors senesce over a concentration range of DETA-NO 0.5-0.75mM or GSNO 1mM, showing an exponential increase in senescence until succumbing to cell death as concentrations of DETA-NO increase. This was confirmed by cell morphology, senescence-associated β-galactosidase activity, total β-galactosidase activity, cell proliferation assays and expression of p16INK4a and p21WAF. We investigated whether this effect could be reproduced by cells induced to express iNOS in vitro. Using these tools, we investigated the mechanism of NO-induced senescence looking at soluble guanylate cyclase activation, increased generation of ROS, protein transnitrosation and glutathione depletion. We investigated ways in which nitric oxide-induced senescence could be circumvented by using antioxidants (NAC, selenomethionine, uric acid), activating AMP kinase with metformin and AICAR and reducing protein transnitrosation by exposing the cells to cold light. We used pharmacological means to mimic lysosomal dysfunction and to stimulate autophagy. My findings show that nitric oxide can cause senescence in vascular cells by a mechanism that involves protein transnitrosation. The effect of nitric oxide on senescence is independent of ROS generation, AMP kinase activation, soluble guanylate cyclase activation and glutathione depletion. My findings may have important implications in vascular disease, particularly in cases of short periods of high nitric oxide production, such as sepsis, but also in the case of low-grade chronic inflammation such as that seen in atherosclerosis. However, their physiological relevance needs to be confirmed. The pathway by which protein transnitrosation induces senescence has yet to be fully elucidated.

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