Abstract-A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4Ј-hydroxy-3Јmethoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems. Key Words: apocynin Ⅲ NADPH oxidase Ⅲ Nox1 Ⅲ Nox4 Ⅲ leukocytes Ⅲ reactive oxygen species R eactive oxygen species (ROS) have a strong impact on vascular homeostasis. 1 ROS originate from several sources, including xanthine oxidase, cytochrome P450 monooxygenases, mitochondria, and uncoupled NO synthase, as well as from the proteins of the Nox family, the NADPH oxidases. The identification of the individual contribution of these generator systems to oxidative burden has been a focus of an impressive amount of publications. The motivation of these studies is that a site-directed ROS lowering therapy to inhibit individual generator systems should be superior to the presently unsuccessful approaches with antioxidants in preventing ROS-dependent cardiovascular diseases. 2 Although it is generally appreciated that molecular techniques involving antisense oligonucleotides, small-interfering RNA, or transgenic animals are to be preferred to pharmacological inhibitors to characterize the contribution of individual ROS generator systems, the latter studies still represent the majority of scientific contributions in the field.Different from N...

Objective-Insulin promotes differentiation of preadipocytes into adipocytes. Insulin also stimulates reactive oxygen species (ROS) production, and the NADPH oxidases Nox1 and Nox4 are important sources of ROS. We determined in human and mouse preadipocytes whether Nox proteins contribute to ROS formation and differentiation in response to insulin. Methods and Results-The expression of Nox1 and Nox4 was increased during insulin-induced differentiation, and insulin increased ROS production. SiRNA against Nox4 but not Nox1 inhibited insulin-induced differentiation and ROS production but promoted proliferation. Nox4 overexpression yielded the opposite effect. As observed by siRNA and overexpression, Nox4 controlled the expression of MAP kinase phosphatase-1 (MKP-1), which reduces insulin-induced ERK1/2 activation. Consequently, downregulation of Nox4 promoted ERK1/2 signaling: Proliferation was increased and through phosphorylation of the inhibitory site serine612, ERK1/2 inhibited the activation of the insulin-receptor substrate-1 (IRS-1) and thereby prevented differentiation in response to insulin. Inhibition of ERK1/2 or overexpression of MPK-1 promoted insulin-induced differentiation. Accordingly, insulin-induced proliferation was enhanced by siRNA against MKP-1, whereas inhibition of ERK1/2 or overexpression of MKP-1 attenuated proliferation. Key Words: oxidative stress Ⅲ superoxide Ⅲ NADPH oxidase Ⅲ differentiation I nsulin is a major adipogen. It stimulates the uptake of glucose, which is then converted into triglycerides and stored in fat droplets leading to adipocyte maturation; insulin also promotes the differentiation of preadipocytes in the stroma of the adipose tissue into adipocytes. 1 Although paracrine autacoids as well as transcription factors and signaling cascades have been identified to be involved in adipocyte differentiation and insulin signaling, these complex processes are still incompletely understood. 2 Insulin is also known to acutely increase the reactive oxygen species (ROS) production in adipocytes, 3 but the enzymes responsible for ROS formation in these cells have not been extensively characterized. Moreover, it is unknown whether ROS are required for insulin-induced differentiation. Conclusions-Nox4Besides mitochondria, the Nox family of NADPH oxidases is considered the most important source of ROS in the body. 4 It is accepted that Nox1-and Nox2-dependent ROS formation requires activation of the proteins by cytosolic activators, whereas Nox4 is constitutively active and independent of activator proteins. 4,5 It is therefore assumed that Nox1 and -2 mediate short-term effects, whereas Nox4 is responsible for long-lasting events such as controlling cell cycle progression and proliferation. 6 We hypothesize that insulin elicits a prolonged increase in ROS formation, which is mediated by an increase in the expression of constitutively active Nox4. Moreover, we investigated whether or not this induction of Nox4 is required for long-term processes in response to insulin, such as prolifer...

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of β-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, β-cell–specific knockout mice (NOX4βKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4βKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause β-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect–plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta. Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect–plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii. In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

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Large bone defects often pose major difficulties in orthopaedic surgery. The application of long-term cultured stem cells combined with a scaffold lead to a significant improvement of bone healing in recent experiments but is strongly restricted by European Union law. Bone marrow mononuclear cells (BMC), however, can be isolated and transplanted within a few hours and have been proven effective in experimental models of bone healing. The effectivity of the BMC-supported therapy might be influenced by the type of scaffold. Hence, we compared three different scaffolds serving as a carrier for BMC in a rat femoral critical size defect with regard to the osteogenic activity in the defect zone. Human demineralized bone matrix (DBM), bovine cancellous bone hydroxyapatite ceramic (BS), or β-tricalcium phosphate (β-TCP) were seeded with human BMC and hereafter implanted into critically sized bone defects of male athymic nude rats. Autologous bone served as a control. Gene activity was measured after 1 week, and bone formation was analysed histologically and radiologically after 8 weeks. Generally, regenerative gene expression (BMP2, RUNX2, VEGF, SDF-1, and RANKL) as well as bony bridging and callus formation was observed to be most pronounced in defects filled with autologous bone, followed in descending order by DBM, β-TCP, and BS. Although DBM was superior in most aspects of bone regeneration analysed in comparison to β-TCP and BS, the level of autologous bone could not be attained.

The Masquelet technique for the treatment of large bone defects is a two‐stage procedure based on an induced membrane. The size of a scaffold is reported to be a critical factor for bone healing response. We therefore aimed to investigate the influence of the granule size of a bone graft substitute on bone marrow derived mononuclear cells (BMC) supported bone healing in combination with the induced membrane. We compared three different sizes of Herafill® granules (Heraeus Medical GmbH, Wehrheim) with or without BMC in vivo in a rat femoral critical size defect. A 10 mm defect was made in 126 rats and a membrane induced by a PMMA‐spacer. After 3 weeks, the spacer was taken out and membrane filled with different granule sizes. After 8 weeks femurs were taken for radiological, biomechanical, histological, and immunohistochemical analysis. Further, whole blood of the rat was incubated with granules and expression of 29 peptide mediators was assessed. Smallest granules showed significantly improved bone healing compared to larger granules, which however did not lead to an increased biomechanical stability in the defect zone. Small granules lead to an increased accumulation of macrophages in situ which could be assigned to the inflammatory subtype M1 by majority. Increased release of chemotactic respectively proangiogenic active factors in vitro compared to syngenic bone and beta‐TCP was observed. Granule size of the bone graft substitute Herafill® has significant impact on bone healing of a critical size defect in combination with Masquelet's technique in terms of bone formation and inflammatory potential.

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