Nox2 oxidase in the vasculature: novel mechanisms of regulation

The Nox2-containing NADPH oxidase enzyme is expressed in a broad range of cell types throughout the body and is generally considered to be the primary source of superoxide within the vasculature under both physiological and pathological conditions. Nox2 oxidase has been directly implicated in endothelial dysfunction, hypertension, vascular inflammation, stroke, diabetes, and atherosclerosis, and thus, it is often considered as a marker of pathology within the vascular system. Conversely, endogenous nitric oxide (NO) is often measured as a marker of vascular integrity, due to its vast number of protective properties. Accordingly, a loss of NO bioavailability is linked to the development of many of the same vascular pathologies that are associated with increased Nox2 oxidase activity. Thus, this thesis examined the hypothesis that endogenous NO regulates Nox2 oxidase-dependent superoxide production within the vasculature to suppress oxidative stress and inflammation. Using a mouse model of chronic NO inhibition, we observed that Nox2 oxidase-dependent superoxide production and vascular inflammation is significantly increased in the aorta of these mice. We subsequently found that the underlying oxidative stress and proinflammatory vascular phenotype could be prevented by suppressing Nox2 oxidase activity through a p47phox-dependent mechanism. These results showed for the first time that endogenous NO might protect the artery wall from developing occlusive atherosclerotic plaques by inhibiting Nox2 oxidase activity through the direct regulation of p47phox expression. Moreover, they provide new insight into the complex mechanisms of NO-dependent vasoprotection and highlight Nox2 oxidase as a potential therapeutic target to treat atherosclerosis. Atherosclerosis is characterised by the formation of macrophage-rich lesions within the blood vessel wall, which can either result in vessel occlusion and reduced blood flow to vital organs or to plaque rupture leading to heart attacks and strokes. It has been shown that macrophage depletion or suppression of Nox2 oxidase-derived superoxide production, dramatically impairs the development of atherosclerotic lesions. In this thesis we examined the macrophage Nox2 oxidase enzyme system in detail and consequently identified for the first time a truncated Nox2 protein specifically expressed in these cells. The protein appears to be regulated during differentiation from the monocyte to the macrophage and is observed within blood vessels of mice with established atherosclerosis. Furthermore, we identified a novel mRNA transcript within various macrophage populations, which shares significant sequence homology with Nox2. We demonstrate that knockdown of this short transcript with siRNA significantly reduces the oxidative burst capacity of the macrophage and that this mRNA encodes an alternatively spliced Nox2 protein, specific to the macrophage, which regulates superoxide production. Furthermore, as the thesis evolved we made two additional interesting observations regarding the regulation of superoxide production. The first observation we made showed that basal superoxide levels are significantly reduced in the aorta of mice genetically deficient in eNOS. We suggest that this observation is the result of a compensatory upregulation in nNOS protein expression and activity which either scavenges vascular superoxide or inhibits its production. The second observation is that Nox2 oxidase is a quantitatively significant source of superoxide under basal conditions in the mouse trachea. These observations suggest that Nox2 oxidase in epithelial cells of the trachea may be implicated in the oxidative stress associated with airway disorders such as asthma and influenza infection. In conclusion, the observations made in this thesis address a number of key gaps in our understanding of the post-transcriptional and post-translational regulation of Nox2 oxidase activity within the vasculature and inflammatory cells such as macrophages. Excitingly, the novel protein and mRNA variants of Nox2 identified represent novel potential drug targets for the treatment of vascular disease and the foundation of future studies in this emerging area of research