Oliver Löwe

The NADPH oxidase Nox4 has anti-atherosclerotic functions

AIMS Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than [Formula: see text] . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme. METHODS AND RESULTS Tamoxifen-induced Nox4-knockout mice were crossed with ApoE⁻/⁻ mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface. CONCLUSIONS The H2O2-producing NADPH oxidase Nox4 is an endogenous anti-atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects.

A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis

Necroptosis is a form of programmed cell death that critically depends on RIP3 and MLKL. However, the contribution of mitochondria to necroptosis is still poorly understood. In the present study, we discovered that mitochondrial perturbations play a critical role in Smac mimetic/Dexamethasone (Dexa)-induced necroptosis independently of death receptor ligands. We demonstrate that the Smac mimetic BV6 and Dexa cooperate to trigger necroptotic cell death in acute lymphoblastic leukemia (ALL) cells that are deficient in caspase activation due to absent caspase-8 expression or pharmacological inhibition by the caspase inhibitor zVAD.fmk, since genetic silencing or pharmacological inhibition of RIP3 or MLKL significantly rescue BV6/Dexa-induced necroptosis. In addition, RIP3 or MLKL knockout mouse embryonic fibroblasts (MEFs) are protected from BV6/Dexa/zVAD.fmk-induced cell death. In contrast, antagonistic antibodies against the death receptor ligands TNFα, TRAIL or CD95 ligand fail to rescue BV6/Dexa-triggered cell death. Kinetic studies revealed that prior to cell death BV6/Dexa treatment causes hyperpolarization of the mitochondrial membrane potential (MMP) followed by loss of MMP, reactive oxygen species (ROS) production, Bak activation and disruption of mitochondrial respiration. Importantly, knockdown of Bak significantly reduces BV6/Dexa-induced loss of MMP and delays cell death, but not ROS production, whereas ROS scavengers attenuate Bak activation, indicating that ROS production occurs upstream of BV6/Dexa-mediated Bak activation. Consistently, BV6/Dexa treatment causes oxidative thiol modifications of Bak protein. Intriguingly, knockdown or knockout of RIP3 or MLKL protect ALL cells or MEFs from BV6/Dexa-induced ROS production, Bak activation, drop of MMP and disruption of mitochondrial respiration, demonstrating that these mitochondrial events depend on RIP3 and MLKL. Thus, mitochondria might serve as an amplification step in BV6/Dexa-induced necroptosis. These findings provide new insights into the role of mitochondrial dysfunctions during necroptosis and have important implications for the development of novel treatment approaches to overcome apoptosis resistance in ALL.

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays

Abstract Measuring NADPH oxidase (Nox)‐derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH‐stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1‐Nox2‐Nox4‐/‐) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH‐stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH‐stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH‐stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH‐dependent signal in assays utilizing lucigenin, L‐012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR‐/‐) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR‐/‐ abolished the signal in NADPH‐stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH‐dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays. Graphical abstract Figure. No Caption available. HighlightsNox activity of intact cells could not be recapitulated in membranes treated with NADPH.Proteomics of membranes show P450 reductase as source of NADPH‐dependent signal.Microsomes overexpressing Cytochrome P450 system produce a NADPH‐dependent signal.Knockout of P450 reductase (CRISPR/Cas9) abolished lucigenin signal in HEK cell membranes.Knockout of POR but not p22phox abolishes the basal and Angiotensin II‐stimulated NADPH‐dependent signal in SMC membranes.

The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype

Objective—Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. Approach and Results—A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1−/− when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1−/− lung endothelial cells (LECs) and a more tip-cell–like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1−/− LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the &agr;-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1−/− LECs when compared with wild-type LECs. Conclusions—NoxO1 stimulates &agr;-secretase activity probably through reactive oxygen species–mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity

The NADPH oxidases are important transmembrane proteins producing reactive oxygen species (ROS). Within the Nox family, different modes of activation can be discriminated. Nox1-3 are dependent on different cytosolic subunits, Nox4 seems to be constitutively active and Nox5 is directly activated by calcium. With the exception of Nox5, all Nox family members are thought to depend on the small transmembrane protein p22phox. With the discovery of the CRISPR/Cas9-system, a tool to alter genomic DNA sequences has become available. So far, this method has not been widely used in the redox community. On such basis, we decided to study the requirement of p22phox in the Nox complex using CRISPR/Cas9-mediated knockout. Knockout of the gene of p22phox, CYBA, led to an ablation of activity of Nox4 and Nox1 but not of Nox5. Production of hydrogen peroxide or superoxide after knockout could be rescued with either human or rat p22phox, but not with the DUOX-maturation factors DUOXA1/A2. Furthermore, different mutations of p22phox were studied regarding the influence on Nox4-dependent H2O2 production. P22phox Q130* and Y121H affected maturation and activity of Nox4. Hence, Nox5-dependent O2•− production is independent of p22phox, but native p22phox is needed for maturation of Nox4 and production of H2O2.