The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis

Hypoxia-inducible Factor-1α Stabilization in Nonhypoxic Conditions: Role of Oxidation and Intracellular Ascorbate Depletion

Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes induced under low oxygen conditions. Under normal oxygen conditions, HIF-1α, the active subunit of HIF-1, is hydroxylated on proline residues by specific HIF prolyl-hydroxylases, leading to ubiquitination and degradation by the proteasome. In hypoxia, hydroxylation and ubiquitination are blocked and HIF-1α accumulates in cells. Recent studies have shown that in normal oxygen conditions G-protein–coupled receptor agonists, including angiotensin (Ang) II and thrombin, potently induce and activate HIF-1 in vascular smooth muscle cells. The current study identifies HIF-1α protein stabilization as a key mechanism for HIF-1 induction by Ang II. We show that hydroxylation on proline 402 is altered by Ang II, decreasing pVHL binding to HIF-1α and allowing HIF-1α protein to escape subsequent ubiquitination and degradation mechanisms. We show that HIF-1α stability is mediated through the Ang II–mediated generation of hydrogen peroxide and a subsequent decrease in ascorbate levels, leading to decreased HIF prolyl-hydroxylase activity and HIF-1α stabilization. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation and will lead to the elucidation of the importance of HIF-1 in different Ang II–related cell responses