Katrin Schröder

Publications of Katrin Schröder

• Leptin Potentiates Endothelium-Dependent Relaxation by Inducing Endothelial Expression of Neuronal NO Synthase.

  Authors: Sebastian Benkhoff, Annemarieke E Loot, Ina Pierson, Adrian Sturza, Karin Kohlstedt, Ingrid Fleming, Hiroaki Shimokawa, Olaf Grisk, Ralf P Brandes, Katrin Schröder

  Arteriosclerosis, thrombosis, and vascular biology. 05/2012;

  OBJECTIVE: Obesity is associated with hyperleptinemia but it is not clear whether leptin protects vascular function or promotes dysfunction. We therefore studied the consequences of hyperleptinemiaOBJECTIVE: Obesity is associated with hyperleptinemia but it is not clear whether leptin protects vascular function or promotes dysfunction. We therefore studied the consequences of hyperleptinemia in lean mice. METHODS AND RESULTS: Wild-type and endothelial NO synthase (eNOS)(-/-) mice were infused with leptin (0.4 mg/kg per day, 7 days), and endothelium-dependent relaxation was studied in aortic segments. Leptin had no effect on acetylcholine-induced endothelium-dependent relaxation in normal wild-type mice but restored endothelium-dependent relaxation in wild-type mice treated with angiotensin II (0.7 mg/kg per day, 7 days) to induce endothelial dysfunction. Leptin also sensitized aortae from eNOS(-/-) mice to acetylcholine, an effect blocked by neuronal NOS (nNOS) inhibition and not observed in eNOS-nNOS double(-/-) mice. Consistent with these findings, leptin induced nNOS expression in murine and human vessels and human endothelial but not smooth muscle cells. Aortic nNOS induction was also induced in mice by a high-fat diet. Mechanistically, leptin increased endothelial Janus kinase 2 and signal transducer and activator of transcription 3 phosphorylation, and inhibition of Janus kinase 2 prevented nNOS induction in cultured cells and leptin-induced relaxations in eNOS(-/-) mice. CONCLUSIONS: Leptin induces endothelial nNOS expression, which compensates, in part, for a lack of NO production by eNOS to maintain endothelium-dependent relaxation.

• Nox4 Is a Protective Reactive Oxygen Species Generating Vascular NADPH Oxidase.

  Authors: Katrin Schröder, Min Zhang, Sebastian Benkhoff, Anja Mieth, Rainer Pliquett, Judith Kosowski, Christoph Kruse, Peter Lüdike, U Ruth Michaelis, Norbert Weissmann, Stefanie Dimmeler, Ajay M Shah, Ralf P Brandes

  Circulation research. 03/2012;

  Rationale:The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction.Objective:We determined the function of Nox4 inRationale:The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction.Objective:We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4(-/-) mice.Methods and Results:Nox4 was highly expressed in the endothelium and contributed to H(2)O(2) formation. Nox4(-/-) mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4(-/-) lung endothelial cells (LECs) was attenuated and restored by low concentrations of H(2)O(2,) whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs.Conclusion:Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.

• The Nox family of NADPH oxidases: friend or foe of the vascular system?

  Authors: Ina Takac, Katrin Schröder, Ralf P Brandes

  Current hypertension reports. 11/2011; 14(1):70-8.

  NADPH (nicotinamide adenine dinucleotide phosphate) oxidases are important sources of reactive oxygen species (ROS). In the vascular system, ROS can have both beneficial and detrimental effects.NADPH (nicotinamide adenine dinucleotide phosphate) oxidases are important sources of reactive oxygen species (ROS). In the vascular system, ROS can have both beneficial and detrimental effects. Under physiologic conditions, ROS are involved in signaling pathways that regulate vascular tone as well as cellular processes like proliferation, migration and differentiation. However, high doses of ROS, which are produced after induction or activation of NADPH oxidases in response to cardiovascular risk factors and inflammation, contribute to the development of endothelial dysfunction and vascular disease. In vascular cells, the NADPH oxidase isoforms Nox1, Nox2, Nox4, and Nox5 are expressed, which differ in their activity, response to stimuli, and the type of ROS released. This review focuses on the specific role of different NADPH oxidase isoforms in vascular physiology and their potential contributions to vascular diseases.

• No superoxide--no stress?: Nox4, the good NADPH oxidase!

  Authors: Ralf P Brandes, Ina Takac, Katrin Schröder

  Arteriosclerosis, thrombosis, and vascular biology. 06/2011; 31(6):1255-7.

• The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4.

  Authors: Ina Takac, Katrin Schröder, Leilei Zhang, Bernard Lardy, Narayana Anilkumar, J David Lambeth, Ajay M Shah, Francoise Morel, Ralf P Brandes

  The Journal of biological chemistry. 02/2011; 286(15):13304-13.

  In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the proteinIn contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).

• Inhibition of the JAK-2/STAT3 signaling pathway impedes the migratory and invasive potential of human glioblastoma cells.

  Authors: Christian Senft, Maike Priester, Margareth Polacin, Katrin Schröder, Volker Seifert, Donat Kögel, Jakob Weissenberger

  Journal of neuro-oncology. 02/2011; 101(3):393-403.

  The objective of current treatment strategies for glioblastoma (GBM) is cytoreduction. Unfortunately, the deleterious migratory and invasive behavior of glial tumors remains largely unattended. TheThe objective of current treatment strategies for glioblastoma (GBM) is cytoreduction. Unfortunately, the deleterious migratory and invasive behavior of glial tumors remains largely unattended. The transcription factor signal transducer and activator of transcription (STAT) 3 is known to be involved in the development and progression of many different tumor types, including malignant gliomas. Beside other biological effects, STAT3 controls cell proliferation and tissue remodeling, processes common to both wound healing and tumor dissemination. Here, we report on impeded migratory and invasive potential of five different glioblastoma cell lines after treatment with AG490, a pharmacological inhibitor of the upstream STAT3 activator Janus kinase (JAK) 2. STAT3 was constitutively activated in all the cell lines tested, and treatment with AG490 eliminated the biologically active, tyrosine705-phosphorylated form of STAT3 in a dose-dependent fashion, as determined by Western blot analysis. Inhibition of activated STAT3 was paralleled by a decrease in transcriptional expression of the STAT3 target genes MMP-2 and MMP-9, and led to reduced proteolytic activity, as determined by zymography. Accordingly, the migratory behavior of all five GBM cell lines was impeded in monolayer wound-healing assays; invasive capacity in matrigel-coated trans-well assays was also hampered by treatment with AG490. The proliferative activity of the cell lines was also significantly reduced after treatment with AG490. The effects elicited by STAT3 inhibition were observed in both PTEN-expressing and PTEN-deficient cells. Because pharmacological inhibition of the JAK-2/STAT3 signaling pathway affects not only tumor cell proliferation but also the characteristic features of malignant gliomas, i.e. migration and invasion pertinent to invariable tumor recurrence and high morbidity, our findings support the idea that STAT3 is a suitable target in the treatment of brain tumors.

• Hepatocyte growth factor induces a proangiogenic phenotype and mobilizes endothelial progenitor cells by activating Nox2.

  Authors: Katrin Schröder, Susanne Schütz, Isabella Schlöffel, Sina Bätz, Ina Takac, Norbert Weissmann, U Ruth Michaelis, Masamichi Koyanagi, Ralf P Brandes

  Antioxidants & redox signaling. 11/2010; 15(4):915-23.

  Hepatocyte growth factor (HGF) by stimulating the receptor tyrosine kinase c-Met induces angiogenesis and tissue regeneration. HGF has been shown to antagonize the angiotensin II-induced senescenceHepatocyte growth factor (HGF) by stimulating the receptor tyrosine kinase c-Met induces angiogenesis and tissue regeneration. HGF has been shown to antagonize the angiotensin II-induced senescence of endothelial progenitor cells (EPCs), which is mediated by NADPH oxidase-dependent reactive oxygen species (ROS) formation. As growth factors, however, usually require ROS for their signaling, we hypothesized that the proangiogenic effects of HGF require NADPH oxidases and focused on the homolog Nox2, which is most abundantly expressed in EPCs and endothelial cells. Indeed, HGF increased the H(2)O(2) formation in EPCs and human umbilical vein endothelial cells (HUVECs), and this effect was not observed in Nox2-deficient cells. HGF induced the mobilization of EPCs and vascular outgrowth from aortic explants in wild-type (WT) but not Nox2(y/-) mice. HGF also stimulated migration and tube formation in HUVECs, and antisense oligonucleotides against Nox2 prevented this effect. To identify the signal transduction underlying these effects, we focused on the kinases Jak2 and Jnk. In HUVECs, HGF increased the phosphorylation of these in a Nox2-dependent manner as demonstrated by antisense oligonucleotides. Also, the HGF-induced Jak2-dependent activation of a STAT3 reporter construct was attenuated after downregulation of Nox2. Accordingly, the HGF-stimulated tube formation of HUVEC was blocked by inhibitors of Jak2 and Jnk. In vivo treatment with the Jnk inhibitor SP600125 blocked the HGF-induced mobilization of EPCs. Ex vivo, SP600125 blocked HGF-induced migration and tube formation. We conclude that HGF-induced mobilization of EPCs and the proangiogenic effects of the growth factor require a Nox2-dependent ROS-mediated activation of Jak2 and Jnk.

• NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis.

  Authors: Min Zhang, Alison C Brewer, Katrin Schröder, Celio X C Santos, David J Grieve, Minshu Wang, Narayana Anilkumar, Bin Yu, Xuebin Dong, Simon J Walker, Ralf P Brandes, Ajay M Shah

  Proceedings of the National Academy of Sciences of the United States of America. 10/2010; 107(42):18121-6.

  Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses, but the role of different ROSCardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses, but the role of different ROS sources remains unclear. Here we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level, which increases in cardiomyocytes under stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models, but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte hypoxia inducible factor 1 and the release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a unique inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of nonspecific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

• NADPH oxidases in cardiovascular disease.

  Authors: Ralf P Brandes, Norbert Weissmann, Katrin Schröder

  Free radical biology & medicine. 05/2010; 49(5):687-706.

  Reactive oxygen species (ROS) contribute to several aspects of vascular diseases including ischemia-reperfusion injury, scavenging of nitric oxide, or stimulation of inflammation and hypertrophy.Reactive oxygen species (ROS) contribute to several aspects of vascular diseases including ischemia-reperfusion injury, scavenging of nitric oxide, or stimulation of inflammation and hypertrophy. NADPH oxidases of the Nox family are differentially expressed in the cardiovascular system, induced or activated by cardiovascular risk factors and importantly contribute to the oxidative burden of vascular diseases. Moreover, NADPH oxidase-derived ROS are important signaling molecules under physiological conditions. In this article, the current knowledge on NADPH oxidase expression, activation, and signaling in the cardiovascular system as well as the impact of risk factors on the function of these proteins will be reviewed. Finally, the contribution of NADPH oxidases to the predominant cardiovascular diseases will be discussed.

• Nox4 is a novel inducible source of reactive oxygen species in monocytes and macrophages and mediates oxidized low density lipoprotein-induced macrophage death.

  Authors: Chi Fung Lee, Mu Qiao, Katrin Schröder, Qingwei Zhao, Reto Asmis

  Circulation research. 04/2010; 106(9):1489-97.

  The enhanced formation of intracellular reactive oxygen species (ROS) induced by oxidized low-density lipoprotein (OxLDL) promotes macrophage death, a process likely to contribute to the formation ofThe enhanced formation of intracellular reactive oxygen species (ROS) induced by oxidized low-density lipoprotein (OxLDL) promotes macrophage death, a process likely to contribute to the formation of necrotic cores and the progression of atherosclerotic lesions. Yet macrophage deficiency of phagocytic NADPH oxidase (Nox2), the primary source of ROS in macrophages, does not reduce atherosclerotic lesion development in mice. This suggests an as yet unidentified NADPH oxidase may be present in macrophages and responsible for the intracellular ROS formation induced by OxLDL. The aim of this study was to identify the source of intracellular ROS involved in macrophage death. Nox4 was expressed in human monocytes and mature macrophages, and was localized to the endoplasmic reticulum and to defined foci within the nucleus. Nox4 colocalized with p22(phox), and both proteins were upregulated in response to OxLDL stimulation, whereas Nox2/gp91(phox) levels remained unchanged. Induction of Nox4 expression, intracellular ROS formation and macrophage cytotoxicity induced by OxLDL were blocked by MEK1/2 inhibition, but not by inhibitors of p38-MAPK (mitogen-activated protein kinase), JNK (Jun N-terminal kinase), or JAK2 (Janus kinase 2). Small interfering RNA knockdown of Nox4 inhibited both intracellular ROS production and macrophage cytotoxicity induced by OxLDL, whereas Nox4 overexpression enhanced both OxLDL-stimulated ROS formation and macrophage death. Nox4 is a novel source of intracellular ROS in human monocytes and macrophages. Induction of Nox4 by OxLDL is mediated by the MEK1/ERK pathway and required for OxLDL cytotoxicity in human macrophages, implicating monocytic Nox4 in atherogenesis.

• Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature.

  Authors: Katrin Schröder

  Current opinion in pharmacology. 02/2010; 10(2):122-6.

  The family of NADPH oxidases consists of seven members that are all producing reactive oxygen species (ROS). In the cardiovascular systems three NADPH oxidases are expressed: Nox1, Nox2 and Nox4,The family of NADPH oxidases consists of seven members that are all producing reactive oxygen species (ROS). In the cardiovascular systems three NADPH oxidases are expressed: Nox1, Nox2 and Nox4, which all exhibit different physiological functions such as regulating blood pressure, mediating growth factor signaling and controlling cell proliferation. In this article the recent research concerning the specificity and function mediated by NADPH oxidases will be reviewed.

• NADPH oxidases are Responsible for the Failure of Nitric Oxide to Inhibit Migration of Smooth Muscle Cells exposed to High Glucose.

  Authors: Xiaoyong Tong, Katrin Schröder

  Free radical biology & medicine. 10/2009;

  Our previous studies showed that nitric oxide (NO) fails to inhibit migration of smooth muscle cells (SMC) exposed to high glucose (HG) because of oxidation of the most reactive cysteine-674 onOur previous studies showed that nitric oxide (NO) fails to inhibit migration of smooth muscle cells (SMC) exposed to high glucose (HG) because of oxidation of the most reactive cysteine-674 on sarco/endoplasmic reticulum ATPase (SERCA), preventing its S-glutathiolation, thus blocking NO action. This study is to further address the sources of oxidants responsible for the failure of NO to inhibit SMC migration in HG. NADPH oxidases are the major source of reactive oxygen species (ROS) in SMC. We used small interference RNA (siRNA) or dominant-negative adenoviral vectors to target components of NADPH oxidase in order to study their individual roles by measuring serum-induced migration in the presence or absence of NO. In HG, the mRNA levels of Nox1 and Nox4 and the protein level of Nox4 were increased; knocking down Nox1 or Nox4 attenuated the ROS production and restored the inhibition of SMC migration by NO. Blockade of the activation of Rac1 or p47(phox) inhibited serum-induced migration and restored the inhibition of migration by NO. These data indicate that NADPH oxidases are responsible for the failure of NO to inhibit SMC migration caused by HG.

• NADPH Oxidase Nox2 Is Required for Hypoxia-Induced Mobilization of Endothelial Progenitor Cells.

  Authors: Katrin Schröder, Andreas Kohnen, Alexandra Aicher, Elisa Liehn, Tom Büchse, Stefan Stein, Christian Weber, Stefanie Dimmeler, Ralf P Brandes

  Circulation research. 09/2009;

  Rationale: Endothelial progenitor cells (EPCs, defined as sca-1(+)flk(+)lin(-) mononuclear blood cells) contribute to vascular repair. The role of hypoxia and reactive oxygen species (ROS) inRationale: Endothelial progenitor cells (EPCs, defined as sca-1(+)flk(+)lin(-) mononuclear blood cells) contribute to vascular repair. The role of hypoxia and reactive oxygen species (ROS) in mobilization and function of these cells is incompletely understood. Objective: We studied the contribution of the NADPH oxidase Nox2, an important vascular source of ROS in this context. Methods and Results: Hypoxia (10% oxygen) induced the mobilization of EPCs in wild-type (WT) and Nox1 but not in Nox2 knockout (Nox2(y/-)) mice. As erythropoietin (EPO) is known to induce EPC mobilization, we focused on this hormone. EPO induced the mobilization of EPCs in WT and Nox1(y/-) but not Nox2(y/-) animals. Transplantation of bone marrow from Nox2(y/-) mice into WT-mice blocked mobilization in response to hypoxia and EPO, whereas transplantation of WT bone marrow into Nox2(y/-) mice restored mobilization. Reendothelialization of the injured mouse carotid artery was enhanced by hypoxia as well as by EPO, and this effect was not observed in Nox2(y/-) mice or after transplantation of Nox2(y/-) bone marrow. In cultured EPCs from WT but not Nox2(y/-) mice, EPO induced ROS production, migration, and proliferation. EPO signaling involves the STAT5 transcription factor. EPO-induced STAT5-dependent reporter gene expression was absent in Nox2-deficient cells. siRNA against the redox-sensitive phosphatase SHP-2 restored EPO-mediated STAT5 induction and inhibition of SHP-2 restored EPO-induced migration in Nox2-deficient cells Conclusions: We conclude that Nox2-derived ROS inactivate SHP-2 and thereby facilitate EPO signaling in EPCs to promote hypoxia-induced mobilization and vascular repair by these cells.

• Nox4 Acts as a Switch Between Differentiation and Proliferation in Preadipocytes.

  Authors: Katrin Schröder, Katalin Wandzioch, Ina Helmcke, Ralf P Brandes

  Arteriosclerosis, thrombosis, and vascular biology. 01/2009;

  OBJECTIVE: Insulin promotes differentiation of preadipocytes into adipocytes. Insulin also stimulates reactive oxygen species (ROS) production, and the NADPH oxidases Nox1 and Nox4 are importantOBJECTIVE: 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. CONCLUSIONS: Nox4 acts as a switch from insulin-induced proliferation to differentiation by controlling MKP-1 expression, which limits ERK1/2 signaling.

• Identification of structural elements in Nox1 and Nox4 controlling localization and activity.

  Authors: Ina Helmcke, Sabine Heumüller, Ritva Tikkanen, Katrin Schröder, Ralf P Brandes

  Antioxidants & redox signaling. 01/2009;

  Nox NADPH oxidases differ in their mode of activation, subcellular localization and physiological function. Nox1 releases superoxide anions (O2-) and depends on cytosolic activator proteins, whereasNox NADPH oxidases differ in their mode of activation, subcellular localization and physiological function. Nox1 releases superoxide anions (O2-) and depends on cytosolic activator proteins, whereas Nox4 extracellularly releases hydrogen peroxide (H2O2) and its activity does not require cotransfection of additional proteins. We constructed chimeric proteins consisting of Nox1 and Nox4 which were expressed in HEK293 cells. When the cytosolic tail of Nox4 was fused with the transmembrane part of Nox1, Nox1 became constitutively active. The reciprocal construct was inactive, suggesting that cytosolic subunit-dependent activation requires elements in the transmembrane loops. By TIRF-microscopy, Nox1 was observed in the plasma membrane, whereas Nox4 co-localized with proteins of the endoplasmic reticulum. Fusion proteins of Myc and Nox revealed that the N-terminal part of Nox1 but not Nox4 is cleaved. When the potential signal peptide of Nox4 was inserted into Nox1, plasma membrane localization was lost and the protein was retained in vesicle-like-structures below the plasma membrane. The potential signal peptide of Nox1 failed to translocate Nox4 to the plasma membrane but switched the extracellularly detectable ROS from H2O2 to O2-. Thus, the very N-terminal part of Nox proteins determines subcellular localization and the ROS type released, whereas the cytosolic tail regulates activity.

• Differential vascular functions of Nox family NADPH oxidases.

  Authors: Ralf P Brandes, Katrin Schröder

  Current opinion in lipidology. 11/2008; 19(5):513-8.

  PURPOSE OF REVIEW: Reactive oxygen species have been implicated in the initiation and progression of atherosclerosis. Reactive oxygen species can oxidize lipoproteins, limit the vascular availabilityPURPOSE OF REVIEW: Reactive oxygen species have been implicated in the initiation and progression of atherosclerosis. Reactive oxygen species can oxidize lipoproteins, limit the vascular availability of antiatherosclerotic nitric oxide and promote vascular expression of cytokines and adhesion molecules. Nox proteins of the NADPH oxidase family are prominent sources of vascular reactive oxygen species, and Nox protein-dependent reactive oxygen species production has been linked to atherogenesis. Recently, significant progress has been made in the understanding of differences among the Nox proteins. RECENT FINDINGS: Nox proteins exhibit cell-specific expression patterns and divergent molecular mechanisms controlling activity have been identified for individual Nox proteins. These aspects may relate to cellular activation, differentiation, proliferation, angiogenesis and gene expression, and may also be modulated by the functional states of the vessel such as endothelial dysfunction: in quiescent vessels, Nox proteins contribute to signal transduction and to the physiological responses to growth factors such as vascular endothelial growth factor or thrombin. Excessive Nox-dependent reactive oxygen species formation in vascular disease such as hyperlipidemia or diabetes, however, largely contributes to vascular dysfunction resulting in defective angiogenesis and inflammatory activation. SUMMARY: Reactive oxygen species, specifically generated by individual Nox proteins, act as secondary messengers. Selective inhibition of Nox proteins might be a novel approach to prevent and treat cardiovascular diseases.

• Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

  Authors: Sabine Heumüller, Sven Wind, Eduardo Barbosa-Sicard, Harald H H W Schmidt, Rudi Busse, Katrin Schröder, Ralf P Brandes

  Hypertension. 03/2008; 51(2):211-7.

  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, reliedA 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 (4'-hydroxy-3'methoxyacetophenone). 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.

• Composition and functions of vascular nicotinamide adenine dinucleotide phosphate oxidases.

  Authors: Ralf P Brandes, Katrin Schröder

  Trends in cardiovascular medicine. 02/2008; 18(1):15-9.

  Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species (ROS) and are expressed in at least three different homologues in the vasculature. TheNicotinamide adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species (ROS) and are expressed in at least three different homologues in the vasculature. The enzymes consist of a membrane complex of one of the large catalytically active Nox proteins and p22phox and different cytosolic subunits. Reactive oxygen species formation by the nicotinamide adenine dinucleotide phosphate oxidases Nox1 and Nox2 in arteries is a consequence of an activation of the enzymes by different stimuli such as growth factors, cytokines, and cardiovascular risk factors (cigarette smoke, high blood pressure, oxidized lipids). Nox4, in contrast, is constitutively active, and therefore, ROS formation by this enzyme is controlled on the expression level of the protein. The negative vascular effects of ROS, such as endothelial dysfunction, vascular hypertrophy, aneurysm formation, and inflammatory activation, appear to be the consequence of an activation of Nox1 and Nox2. Nox4, in contrast, potentially elicits positive effects because it promotes differentiation and reduces proliferation of cells. Consequently, selective pharmacologic inhibition of Nox proteins has a potential to interfere with cardiovascular disease initiation and progression.

• Nox1 mediates basic fibroblast growth factor-induced migration of vascular smooth muscle cells.

  Authors: Katrin Schröder, Ina Helmcke, Katalin Palfi, Karl-Heinz Krause, Rudi Busse, Ralf P Brandes

  Arteriosclerosis, thrombosis, and vascular biology. 09/2007; 27(8):1736-43.

  OBJECTIVE: Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role ofOBJECTIVE: Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role of ROS for SMC migration. METHODS AND RESULTS: bFGF rapidly increased rat SMC ROS formation and migration through pathways sensitive to inhibition of NADPH oxidases, PI3-kinase, protein kinase C, and Rac-1. SiRNA directed against the NADPH oxidase Nox4 impaired basal but not bFGF-induced ROS formation and did not affect migration. In contrast, siRNA against Nox1 blocked the agonist-induced ROS generation as well as the bFGF-induced migration. Agonist-induced migration was also attenuated in SMC derived from Nox1 y/- mice and transduction of Nox1 restored normal migration. Likewise, SMC outgrowth in response to bFGF was attenuated in aortic segments from Nox1 y/- mice as compared with Nox1 y/+ mice. bFGF activated JNK but not Src in a Nox1-dependent manner. Consequently, phosphorylation of the adaptor protein paxillin, which is central for migration and secretion of matrix-metalloproteinases, were dependent on Nox1 as well as JNK but not Src. CONCLUSIONS: These data demonstrate that bFGF activates the Nox1-containing NADPH oxidase and that bFGF through a pathway involving ROS and JNK stimulates SMC migration.

• Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet.

  Authors: Katrin Schröder, Carmine Vecchione, Oliver Jung, Judith G Schreiber, Ronit Shiri-Sverdlov, Patrick J van Gorp, Rudi Busse, Ralf P Brandes

  Free radical biology & medicine. 12/2006; 41(9):1353-60.

  Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO isHyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis. ApoE(-/-) mice were fed a Western-type (WD) or control diet. In subgroups, tungsten (700 mg/L) was administered to inhibit XO. XO is a secreted enzyme which is formed in the liver as xanthine dehydrogenase (XDH) and binds to the vascular endothelium. High expression of XDH was found in the liver and WD increased liver XDH mRNA and XDH protein expression. WD induced the conversion of XDH to the radical-forming XO. Moreover, WD increased the hepatic expression of CD40, demonstrating activation of hepatic cells. Aortic tissue of ApoE(-/-) mice fed a WD for 6 months exhibited marked atherosclerosis, attenuated endothelium-dependent relaxation to acetylcholine, increased vascular oxidative stress, and mRNA expression of the chemokine KC. Tungsten treatment had no effect on plasma lipids but lowered the plasma XO activity. In animals fed a control diet, tungsten had no effect on radical formation, endothelial function, or atherosclerosis development. In mice fed a WD, however tungsten attenuated the vascular superoxide anion formation, prevented endothelial dysfunction, and attenuated KC mRNA expression. Most importantly, tungsten treatment largely prevented the development of atherosclerosis in the aorta of ApoE(-/-) mice on WD. Therefore, tungsten, potentially via the inhibition of XO, prevents the development of endothelial dysfunction and atherosclerosis in ApoE(-/-) mice on WD.