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Oxidative stress in endothelial cells of patients with coronary artery disease

Mackenzie, Ruth M. (2010) Oxidative stress in endothelial cells of patients with coronary artery disease. PhD thesis, University of Glasgow.

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Abstract

There is increasing evidence to support a role for vascular oxidative stress in the development of endothelial dysfunction and coronary artery disease (CAD). The prevalence of CAD in patients with type 2 diabetes (T2D) is more than ten times greater than in the general population and CAD patients with T2D display poorer clinical outcomes than those without. Previous investigations carried out within this laboratory have demonstrated endothelial dysfunction and elevated superoxide (O2-) production throughout the vessel wall in saphenous veins from patients with advanced CAD. However, the molecular basis for these findings was not investigated, nor was the effect of T2D. Consequently, the aim of this study was to investigate a number of molecular determinants of oxidative stress in primary human saphenous vein endothelial cells (HSVECs) from CAD patients both with and without T2D, the hypothesis being that oxidative stress leads to endothelial dysfunction in CAD and is exacerbated in those patients with T2D. Initial experiments confirmed the findings of previous studies, revealing significantly elevated O2- levels and impaired endothelium-dependent relaxation in portions of saphenous vein from patients with CAD related to those from healthy control subjects. In addition, results demonstrated more severe endothelial dysfunction in vessels from CAD patients with T2D relative to those CAD patients without this additional cardiovascular risk factor. Attempts to characterise cellular sources of elevated O2- production in the endothelium of patients with CAD revealed that while HSVEC endothelial nitric oxide synthase (eNOS) is not a major source of O2- generation in CAD, nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) oxidase contributes to excess O2- production in the endothelium of patients with this cardiovascular disease. Further cellular investigation revealed a significant increase in expression of mitochondrial superoxide dismutase (SOD2) in HSVECs from CAD patients, in particular those with T2D. Mitochondrially-derived reactive oxygen species (mtROS) are reported to contribute to oxidative stress and endothelial dysfunction in CAD and T2D and, as such, this increase in SOD2 expression is likely to represent an adaptive response to elevated mitochondrial O2- production. In addition, analysis of gene expression microarray data revealed differential expression of a number of genes potentially linked to mitochondrial function, serving to support a key role for mitochondrial dysfunction in the pathogenesis of CAD and T2D. Additional molecular studies focused on AMP-activated protein kinase (AMPK). Proposed as a candidate target for therapeutic intervention in endothelial dysfunction, AMPK stimulates eNOS, leading to nitric oxide (NO) production in cultured endothelial cells. Furthermore, it has recently been reported that endothelial AMPK is activated in a mtROS-mediated manner. With SOD2 expression data indicating an increase in mtROS production in the endothelium of patients with CAD, AMPK activity was investigated and compared in HSVECs isolated from these patients and control subjects. AMPK activity was significantly greater in cells from patients with CAD, despite no change in activity of upstream kinases, LKB1 and Ca2+/calmodulin-dependent kinase kinase (CaMKK). On stratifying CAD patients according to the presence of T2D, AMPK activity was found to be significantly increased in the endothelium of those patients with CAD and T2D as compared to those with CAD alone. Given the potentially elevated levels of mtROS production in the endothelium of patients with CAD, it seemed likely that endothelial AMPK activation in CAD patients was occurring in an mtROS-mediated manner and that this activation was enhanced in those CAD patients with T2D who display more severe endothelial dysfunction and increased SOD2 expression, consistent with increased mtROS production. In order to test this hypothesis, HSVECs isolated from vessels of CAD patients were treated with the mitochondria-targeted antioxidant, MitoQ10. Results demonstrated a significant decrease in AMPK activation in cells from those CAD patients with T2D on treatment with MitoQ10. The same effect was not seen in cells from CAD patients without T2D. These findings indicate that AMPK is activated in a mtROS-mediated manner in endothelial cells isolated from CAD patients with T2D and suggest a role for the kinase in defence against oxidative stress and endothelial dysfunction in these individuals. In summary, a wide range of molecular techniques have been employed to investigate cellular mechanisms underlying the oxidative stress and endothelial dysfunction associated with CAD. Results suggest mitochondria contribute to the increased O2- production and endothelial dysfunction observed in vessels from CAD patients. In addition, findings indicate a novel, mtROS-mediated activation of AMPK in the endothelium of patients with CAD and T2D. Therefore, mitochondria-targeted antioxidants, used in combination with pharmacological activators of AMPK, may have enhanced potential in prevention and treatment of coronary artery disease in patients with type 2 diabetes.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: coronary artery disease, type 2 diabetes mellitus, endothelium, endothelial dysfunction, oxidative stress, mitochondrial reactive oxygen species (mtROS), AMP-activated protein kinase (AMPK)
Subjects: R Medicine > RC Internal medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Supervisor's Name: Dominiczak, Prof. Anna F. and Miller, Dr. William H.
Date of Award: 2010
Depositing User: Dr Ruth M. Mackenzie
Unique ID: glathesis:2010-1531
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 15 Mar 2010
Last Modified: 10 Dec 2012 13:42
URI: http://theses.gla.ac.uk/id/eprint/1531

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