NADPH Oxidase

Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are associated with conditions such as hypertension, diabetes and hypercholesterolemia and have emerged as key regulators of vascular pathologies (i.e. oxidative stress, endothelial dysfunction, inflammation, remodelling).. Our laboratory is focussed on identifying the major enzymatic sources of ROS at the various stages of vascular disease with a view to developing novel therapeutic strategies that directly target these enzymes.

NADPH oxidases are a family of enzyme complexes whose primary function is to catalyse the transfer of electrons from NADPH to molecular oxygen resulting in the generation of superoxide. This function makes NADPH oxidases unique and distinguishes it from all other oxidases where superoxide production occurs either as a by-product of another oxidative reaction (e.g. the mitochondrial electron transport chain) or from a dysfunctional variant of the enzyme (e.g. uncoupled eNOS, xanthine oxidase).

Figure 1.  Model of NADPH oxidases and their regulatory subunits. Nox1-4 are localized at the membrane associated to the p22phox protein. (A) The activity of Nox1-3 is regulated by several factors. The enzymatic active complex of Nox1 contains the Nox organizer 1 (Noxo1), the Nox activator 1 (Noxa1) and the small GTPase Rac in its active GTP-bound state. The activity of Nox2 (alias gp91phox) depends on p47phox, p67phox and Rac; p40phox can further support its activity. Nox3 is already active in a complex with Noxo1 alone; the function of Rac for Nox3 activity in vivo is not clear. p47phox is binding mainly to phosphatidylinositol (3,4) bisphosphate (PIP2) via its PX domain, whereas Noxo1 binds to monophosphorylated phosphoinositides (PIP), like PI(4)P or PI(5)P. Noxo1β and Noxo1γ are representing the most common splice variants of Noxo1. (B) Nox4 is not regulated by any of the known regulatory subunits. Also the influence of Rac on Nox4 activity is still under discussion. Nox5 is known to be activated by Calcium through binding to its EF-hand motifs resulting in a conformational change and thereby releasing its intramolecular inhibitory binding.

We have shown that during normal physiology, blood vessels express a single isoform of NADPH oxidase (Nox4), which produces low amounts of ROS in a tightly regulated fashion for use in redox-dependent signalling processes. Cytosolic subunits are not associated with Nox4, and H₂O₂ is the major form of ROS generated. In rodent models of hypertension, diabetes and hypercholesterolemia, two additional isoforms of NADPH oxidase (Nox1 & Nox2) are upregulated in the blood vessel wall, possibly even prior to the appearance of clinical symptoms. Upregulation of these Nox isoforms results in a modest, albeit pathologically relevant increase in vascular ROS production, such that endogenous antioxidant systems become overwhelmed. These excess ROS molecules participate in various redox reactions with important vasoprotective biomolecules such as nitric oxide (NO). This not only causes a reduction in NO bioavailability and a loss of many of its protective functions, but also results in the formation of even more powerful oxidants such as peroxynitrite (ONOO^-) and hydroxyl radicals (OH•). Therefore, these ROS are thought to be the major causes of oxidative damage to lipids, proteins and even DNA. In addition, ONOO^- has been shown to oxidatively deplete the essential eNOS cofactor, tetrahydrobiopterin, and to oxidise critical cysteine thiol residues on xanthine dehydrogenase. These actions result in eNOS uncoupling and in the formation of xanthine oxidase, respectively, which effectively switch off the normal catalytic functions of these enzymes and convert them into ROS generating systems. This process whereby upregulation of an initial source of ROS (i.e. Nox1 and Nox2) in early stage disease activates normally dormant sources of ROS (eNOS and xanthine dehydrogenase) in the later stages of pathology is analogous to a kindling-bonfire effect. One of the major strengths of the kindling-bonfire Hypothesis of vascular oxidative stress is that it provides a framework for future diagnostic applications focussed on assessing patients’ oxidative status, and for potential therapeutic strategies for reducing oxidative stress, which are tailored to an individual patient depending on the stage of his/her disease progression. Interestingly, rodents appear to have selectively lost Nox5, while other mammals have one Nox5 gene. In humans, there are at least 5 Nox5 splice variants among one (Nox5 delta) is expressed in human microvascular endothelial cell. This points to a yet unidentified role of Nox5 in human vasculature.  The VDDG will elucidate the relative role of different NOX isoforms in human vascular disease and disease models to develop more specific therapies.