NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives

Abstract

Oxidative stress caused by an excess of reactive oxygen species (ROS) is known to contribute to stroke injury, particularly during reperfusion, and antioxidants targeting this process have resulted in improved outcomes experimentally. Unfortunately these improvements have not been successfully translated to the clinical setting. Targeting the source of oxidative stress may provide a superior therapeutic approach. The NADPH oxidases are a family of enzymes dedicated solely to ROS production and pre-clinical animal studies targeting NADPH oxidases have shown promising results. However there are multiple factors that need to be considered for future drug development: There are several homologues of the catalytic subunit of NADPH oxidase. All have differing physiological roles and may contribute differentially to oxidative damage after stroke. Additionally, the role of ROS in brain repair is largely unexplored, which should be taken into consideration when developing drugs that inhibit specific NADPH oxidases after injury. This article focuses on the current knowledge regarding NADPH oxidase after stroke including in vivo genetic and inhibitor studies. The caution required when interpreting reports of positive outcomes after NADPH oxidase inhibition is also discussed, as effects on long term recovery are yet to be investigated and are likely to affect successful clinical translation.

Figure 1

Diagrammatic representation of the resting and activated forms of the phagocytic NADPH oxidase. The catalytic subunit, gp91phox, along with p22phox makes up flavocytochrome b558, the membrane-associated component of the enzyme. The three phox proteins, p47, p67 and p40 form a cytosolic complex in the resting cell, and upon activation translocate to the membrane, docking with flavocytochrome b558. The small G-protein Rac, in its GDP-bound form, is stabilised by RhoGDI in the resting state and also translocates to the membrane upon activation. When assembled, the enzyme generates the ROS superoxide (O₂⁻) by accepting electrons (e⁻) from cytoplasmic NADPH and donating them to molecular oxygen (O₂).

Figure 2

Simplified schematic illustrating the major pathways for the formation of ROS and reactive nitrogen species (RNS) originating from the Nox/Duox enzymes. Superoxide (O₂⁻) is generated by the Nox enzymes and can be dismutated to hydrogen peroxide (H₂O₂), either spontaneously or catalysed by superoxide dismutase (SOD), or can react with nitric oxide (NO) to form peroxynitrite (ONOO⁻). H₂O₂, generated by the Duox enzymes or by dismutation of O₂⁻ can be scavenged by the antioxidants catalase (CAT) or glutathione peroxidase (GPx) to form water (H₂O) and oxygen (O₂); be partially reduced to generate hydroxyl radical (OH) by the metal (Fe³⁺) catalysed Haber-Weiss and Fenton reactions; or react with chloride in a reaction catalysed by myeloperoxidase (MPO), resulting in formation of hypochlorous acid (HOCl).

Figure 3

Nox2 immunohistochemistry in the stroke affected cortex is associated with vascular sprouting, in addition to inflammatory cells, 7 days after stroke. Immunofluorescent images of von Willebrand factor labelled blood vessels (red; A,D) and Nox2 labelled cells (green; B,E) in the stroke affected cortex 7 days (A–C) and 28 days (B–E) after endothelin-1 induced stroke. Merged images (C,E) reveal angiogenic vessels at 7 days (C) are double labelled with Nox2, suggesting a role for Nox2 in vascular sprouting, an effect that is no longer present 28 days after stroke (E), once vessels have matured. Scale = 100 µm.