AimNADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes.ResultsIn the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice.Innovation and conclusionROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response.

NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces H 2 O 2 which can directly oxidize cysteine residues. With this post-translational modification, activity, stability, localization and protein-protein interactions of the affected protein is altered. Nox4 controls differentiation, cellular homeostasis and prevents inflammation. Therefore, is likely that epigenetic mechanisms contribute to the effects of Nox4. One group of epigenetic modifiers are class IIa histone deacetylases (HDACs). We hypothesize that Nox4-derived H 2 O 2 oxidizes HDACs and analyzed whether HDACs can be differentially oxidized by Nox4. As an artificial system, we utilized HEK293 cells, overexpressing Nox4 in a tetracycline-inducible manner. HDAC4 was oxidized upon Nox4 overexpression. Additionally, Nox4 overexpression increased HDAC4 phosphorylation on Ser632. H 2 O 2 disrupted HDAC4/Mef2A complex, which de-represses Mef2A. In endothelial cells such as HUVECs and HMECs, overexpression of HDAC4 significantly reduced tube formation. Overexpression of a redox insensitive HDAC4 had no effect on endothelial tube formation. Treatment with H 2 O 2 , induction of Nox4 expression by treatment of the cells with TGFβ and co-overexpression of Nox4 not only induced phosphorylation of HDAC4, but also restored the repressive effect of HDAC4 for tube formation, while overexpression of a redox dead mutant of Nox4 did not. Taken together, Nox4 oxidizes HDAC4, increases its phosphorylation, and eventually ensures proper tube formation by endothelial cells.

Objective— PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results— Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47 phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47 phox and PDI and an intramolecular bond between Cys 196 and 378 in p47 phox . PDI catalytic Cys 400 and p47 phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47 phox , whereas PDI mutant did not promote this effect. Mutation of p47 phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47 phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47 phox occurred only in PDI family members with the a′ redox active site. Conclusions— PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.

The differentiation of stem cells into endothelial cells involves the modulation of highly interconnected metabolic and epigenetic processes. Therefore, the differentiation of endothelial cells is a tightly controlled process, which is adjusted at multiple levels, meaning that even the smallest variation can result in major consequences. Reactive oxygen species (ROS) represent a group of second messengers that can interfere with both metabolic and epigenetic processes. Besides their generation by mitochondria, ROS are produced in a controlled manner by the family of NADPH oxidases. The different members of the NADPH oxidase family produce superoxide anions or hydrogen peroxide. Due to the specific sub-cellular localization of the different NADPH oxidases, ROS are produced at diverse sites in the cell, such as the plasma membrane or the endoplasmic reticulum. Once produced, ROS interfere with proteins, lipids, and DNA to modulate intracellular signal cascades. Accordingly, ROS represent a group of readily available and specifically localized modulators of the highly sophisticated signalling network that eventually leads to the differentiation of stem cells into endothelial cells. This review focuses on the role of NADPH oxidases in the differentiation of stem cells into endothelial cells.

We perform the maximal twist of eleven-dimensional supergravity. This twist is partially topological and exists on manifolds of G 2 ×SU (2) holonomy. Our derivation starts with an explicit description of the Batalin-Vilkovisky complex associated to the three-form multiplet in the pure spinor superfield formalism. We then determine the L ∞ module structure of the supersymmetry algebra on the component fields. We twist the theory by modifying the differential of the Batalin-Vilkovisky complex to incorporate the action of a scalar supercharge. We find that the resulting free twisted theory is given by the tensor product of the de Rham and Dolbeault complexes of the respective G 2 and SU (2) holonomy manifolds as conjectured by Costello.