Reactive Oxygen Species in Human Blood Vessels: Sources of Superoxide Production and Effects of Angiotensin II

Berry, Colin (2002) Reactive Oxygen Species in Human Blood Vessels: Sources of Superoxide Production and Effects of Angiotensin II. PhD thesis, University of Glasgow.

Abstract

The purpose of this thesis was to investigate the enzymatic sources of, and stimuli for, reactive oxygen species (ROS) production in human vascular cells and blood vessels. In particular, the possibility that vasoactive hormones, such as angiotensin II (Ang II), might stimulate vascular ROS production was also explored. Internal mammary arteries (IMA) and saphenous veins (SV) were collected at the time of coronary artery bypass surgery. Initial validation studies demonstrated that lucigenin-enhanced chemiluminescence was a sensitive and specific method for quantification of superoxide (02) concentrations in these blood vessels. The enzymatic sources of ROS generation were NAD(P)H oxidase, xanthine oxidase and in some, but not all, patients, nitric oxide synthase. Superoxide production was greater in IMA than in SV, whereas the amount of superoxide dismutase protein was quantitatively similar in these blood vessels. In subsequent studies, treatment of IMA with pharmacological concentrations of Ang II (1 micromolar, 1 nanomolar) for 1 and 4 hours was associated with an increase in O2-production. Treatment of IMA with picomolar, or physiological, concentrations of Ang II tended to increase O2- production. Further studies demonstrated that this was an Ang type 1 (AT1) receptor-dependent, NAD(P)H oxidase-mediated pathway. Furthermore, inhibition of the AT2 receptor did not prevent Ang II-stimulated increase in O2- production in IMA, suggesting that this receptor does not contribute to O2- generation in human arteries. Immunodetection studies for the NAD(P)H oxidase phox subunits were performed in IMA. Using monoclonal antibodies and antisera, p22phox, gp9I phox (or a homologue), p67phox and p47 phox protein subunits were identified within the endothelium, vascular smooth muscle cell layer and adventitia. Xanthine oxidase was identified in the endothelium and adventitial layers of IMA. In this chapter, studies of the effects of Ang II on the abundance of cDNA transcripts of the subunits of NAD(P)H oxidase are reported. In subsequent molecular studies, it was demonstrated that treatment of human vascular cells and intact IMA with 1 micromole of Ang II for 4 hours led to an increase in the abundance of cDNA transcripts of p22phox, which was attenuated by co-treatment with either actinomycin D, an inhibitor of gene transcription, or losartan, an AT1 receptor antagonist. In other studies, the possibility that Ang II-stimulated O2- production might contribute to vascular tone in human subcutaneous resistance arteries (SRA) was investigated. Isometric tension studies failed to demonstrate any positive effect of Ang II on the contractile response of SRA to norepinephrine. One reason for this may be the relatively minor contribution of nitric oxide to the vasorelaxant response of these arteries. An investigation of the effect, if any, of demographic characteristics, risk factors for atherosclerosis, and individual drug therapies, on vascular O2- production in the IMA of 79 patients was also performed. Multivariate analyses demonstrated that increasing age was weakly associated with increased vascular O2- production whereas treatment with an angiotensin converting enzyme inhibitor or AT1 receptor antagonist was independently associated with reduced vascular O2- concentrations. In summary, O2- production is greater in IMA than in SV. Several enzymes capable of generating ROS are distributed throughout the wall of these arteries. NAD(P)H oxidase is a major source of O2- generation. The activity of this enzyme is enhanced by Ang II, through a mechanism which involves and increase in gene transcription and protein synthesis. This may be clinically important as inhibitors of the renin- aldosterone-angiotensin system may reduce vascular O2- production.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: A F Dominiczak
• Keywords: Medicine, Physiology
• Date of Award: 2002
• Depositing User: Enlighten Team
• Unique ID: glathesis:2002-76039
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 17:05
• Last Modified: 19 Nov 2019 17:05
• URI: https://theses.gla.ac.uk/id/eprint/76039