Defining the role(s) of Nox2-NADPH oxidase in the cerebral circulation

Evidence suggests that NADPH oxidase-derived reactive oxygen species (ROS), such as superoxide (O2-) and hydrogen peroxide (H2O2), are important signalling molecules within cerebral arteries. However, if there is an imbalance between ROS production and metabolism oxidative stress can ensue. One of the major consequences of oxidative stress is a decrease in the bioactivity of the vasoprotective molecule nitric oxide (NO•). This thesis examined the role of cerebrovascular Nox2-containing NADPH oxidase in (1) gender-dependent differences in responses to angiotensin II in mice; and (2) excessive ROS production and diminished NO• function in mouse models of hypercholesterolaemia and ischaemic stroke. The major experimental approaches used in this thesis were 1) chemiluminescence and fluorescence techniques for the measurement of ROS production; 2) perfusion myography for the assessment of NO• function in vitro; and 3) western blotting and immunofluorescence for the localization and measurement of various proteins of interest. In Chapter 3, we demonstrate that cerebrovascular ROS production and constrictor responses to angiotensin II are considerably smaller in female versus male wild type (WT) mice. In male Nox2-deficient (Nox2-/-) mice, responses to angiotensin II were attenuated, whereas Nox2 deletion had no effect on responses in female mice. These Nox2-related gender-dependent differences were not associated with differences in Nox2 protein expression or cellular localization. Taken together, these data demonstrate for the first time that these gender-dependent differences in responses to angiotensin II are likely to be due to lower Nox2 activity in females. Another novel finding of Chapter 3 was that Nox2-derived ROS is critical mediator of angiotensin II-induced constrictions in the cerebral circulation of male mice. Therefore, in Chapter 4 we sought to determine the identity of the ROS responsible. Using pharmacological scavengers of O2- and H2O2 we found evidence that H2O2 (or a downstream ROS) mediates constrictions to angiotensin II in the mouse cerebral circulation. In Chapter 5, we found that despite the absence of atherosclerotic lesions, cerebrovascular O2- production was augmented and NO• function was impaired in hypercholesterolaemic apolipoprotein E-deficient (ApoE-/-) mice versus WT mice. However, in Nox2-/-/ApoE-/- mice, cerebrovascular O2- production was not elevated and NO• function was comparable to that in WT mice. Protein expression of Nox2 was not elevated in ApoE-/- mice, however, expression of p47phox (Nox organizer) was 2-fold higher in cerebral arteries from ApoE-/- mice. Therefore, this study demonstrates that excessive O2- production and impaired by NO• function occur in cerebral arteries of hypercholesterolaemic mice, as a consequence of increased activity of Nox2-NADPH oxidase. In Chapter 6, we found that at 24 h after middle cerebral artery occlusion in WT mice, O2- production is augmented and NO• function is impaired in ischaemic versus non-ischaemic middle cerebral arteries (MCA). By contrast, in Nox2-/- mice, cerebrovascular O2- production and NO• function was comparable between ischaemic and non-ischaemic MCA. Thus, Nox2-NADPH oxidase plays a central role in cerebrovascular NO• dysfunction following ischaemia and reperfusion. In summary, the findings of this thesis indicate that Nox2-derived ROS plays a key role in mediating cerebrovascular responses to angiotensin II and in mediating impaired NO• function during hypercholesterolaemia and following stroke. While future work is needed to determine whether similar changes occur in humans and in other vascular diseases, these findings position Nox2-NADPH oxidase as a strong candidate for the excessive ROS production that is thought to lead to oxidative stress associated with a number of vascular diseases. As such, we propose that Nox2-NADPH oxidase may represent a therapeutic target to alleviate the burden of oxidative stress-induced vascular dysfunction