Gregor Muller

Publications of Gregor Muller

• Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells.

  Authors: Claudia Goettsch, Winfried Goettsch, Melanie Brux, Claudia Haschke, Coy Brunssen, Gregor Muller, Stefan R Bornstein, Nicole Duerrschmidt, Andreas H Wagner, Henning Morawietz

  Basic research in cardiology. 03/2011; 106(4):551-61.

  The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex;The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduces endothelial superoxide anion formation and Nox4 expression. In primary human endothelial cells, we identified a 47 bp 5'-untranslated region of Nox4 mRNA by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Cloning and functional analysis of human Nox4 promoter revealed a range between -1,490 and -1,310 bp responsible for flow-dependent downregulation. Mutation of an overlapping antioxidative response element (ARE)-like and Oct-1 binding site at -1,376 bp eliminated shear stress-dependent Nox4 downregulation. Consistent with these observations, electrophoretic mobility shift assays (EMSA) demonstrated an enhanced shear stress-dependent binding of Nox4 oligonucleotide containing the ARE-like/Oct-1 binding site, which could be inhibited by specific antibodies against the transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and octamer transcription factor 1 (Oct-1). Furthermore, shear stress caused the translocation of Nrf2 and Oct-1 from the cytoplasm to the nucleus. Knockdown of Nrf2 by short hairpin RNA (shRNA) increased Nox4 expression twofold, indicating a direct cross-talk between Nrf2 and Nox4. In conclusion, an ARE-like/Oct-1 binding site was noticed to be essential for shear stress-dependent downregulation of Nox4. This novel mechanism may be involved in the flow-dependent downregulation of endothelial superoxide anion formation.

• Impaired vascular function in small resistance arteries of LOX-1 overexpressing mice on high-fat diet.

  Authors: Birgit Eichhorn, Gregor Muller, Anja Leuner, Tatsuya Sawamura, Ursula Ravens, Henning Morawietz

  Cardiovascular research. 05/2009;

  AIMS: LOX-1 is a major vascular receptor for oxidized low-density lipoprotein (oxLDL). In this study, we analysed the impact of LOX-1 overexpression and high dietary fat intake on vascular functionAIMS: LOX-1 is a major vascular receptor for oxidized low-density lipoprotein (oxLDL). In this study, we analysed the impact of LOX-1 overexpression and high dietary fat intake on vascular function in small resistance arteries. METHODS AND RESULTS: Relaxation of mesenteric arteries was measured using a wire myograph. Compared with the control group, mice overexpressing LOX-1 on a high-fat diet (FD) had preserved vascular smooth muscle relaxation, but impaired endothelium-dependent relaxation via NO. Vascular NO availability was decreased by exaggerated formation of reactive oxygen species and decreased endothelial NO synthase expression. Endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation via cytochrome P450 metabolites was increased in LOX-1 + FD animals, but did not completely compensate for the loss of NO. Currents of calcium-activated potassium channels with large conductance (BK(Ca) channels) were measured by the voltage-clamp method. The BK(Ca) current amplitudes were not altered in endothelial cells, but highly increased in vascular smooth muscle cells from resistance arteries of LOX-1-overexpressing mice on FD. BK(Ca) currents were activated by low-dose H(2)O(2) and cytochrome P450 metabolites 11,12-EET and 14,15-EET as EDHF in control mice. CONCLUSION: LOX-1 overexpression and FD caused functional changes in endothelial and vascular smooth muscle cells of small resistance arteries.

• Nitric Oxide, NAD(P)H Oxidase and Atherosclerosis.

  Authors: Gregor Muller, Henning Morawietz

  Antioxidants & redox signaling. 04/2009;

  The endothelial cell layer plays a major role in the development and progression of atherosclerosis. Endothelial NO synthase (eNOS) produces nitric oxide (NO) from L-arginine. NO can rapidly reactThe endothelial cell layer plays a major role in the development and progression of atherosclerosis. Endothelial NO synthase (eNOS) produces nitric oxide (NO) from L-arginine. NO can rapidly react with reactive oxygen species to form peroxynitrite. This reduces NO availability, impairs vasodilatation and mediates proinflammatory and prothrombotic processes such as leukocyte adhesion and platelet aggregation. In the vessel wall, specific NAD(P)H oxidase complexes are major sources of reactive oxygen species. These NAD(P)H oxidases can transfer electrons across membranes to oxygen and generate superoxide anions. The short-lived superoxide anion rapidly dismutates to hydrogen peroxide, which can further increase the production of reactive oxygen species. This can lead to uncoupling of eNOS switching enzymatic activity from NO to superoxide production. This review describes the structure and regulation of different NAD(P)H oxidase complexes. We will also focus on NO/superoxide anion balance as modulated by hemodynamic forces, vasoconstrictors and oxidized low-density lipoprotein. We will then go on to summarize the recent advances defining the role of nitric oxide and NAD(P)H oxidase-derived reactive oxygen species in the development and progression of atherosclerosis. In conclusion, novel mechanisms affecting the vascular NO/superoxide anion balance will allow the development of therapeutic strategies in the treatment of cardiovascular diseases.

• Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells.

  Authors: Claudia Goettsch, Winfried Goettsch, Gregor Muller, Jochen Seebach, Hans-Joachim Schnittler, Henning Morawietz

  Biochemical and biophysical research communications. 02/2009;

  Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCRNicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22(phox) expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 downregulation by shRNA has in contrast to TGF-beta no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22(phox). The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.

• NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells.

  Authors: Nicole Duerrschmidt, Claudia Stielow, Gregor Muller, Patrick J Pagano, Henning Morawietz

  The Journal of physiology. 11/2006; 576(Pt 2):557-67.

  The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-))The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analysed the effect of laminar shear stress on .O(2)(-) formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced .O(2)(-) formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm(-2), 24 h) down-regulated .O(2)(-) formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91(phox) and p47(phox). In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of .O(2)(-) formation, gp91(phox) and p47(phox) expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates .O(2)(-) formation, gp91(phox) and p47(phox) expression in static cultures. In conclusion, our data suggest a transient activation of .O(2)(-) formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91(phox)/p47(phox)-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/.O(2)(-) balance and the vasoprotective potential of physiological levels of laminar shear stress.

• Upregulation of endothelin receptor B in human endothelial cells by low-density lipoproteins.

  Authors: Gregor Muller, Rusan A Catar, Bernd Niemann, Matthias Barton, Lilla Knels, Martina Wendel, Henning Morawietz

  Experimental biology and medicine (Maywood, N.J.). 06/2006; 231(6):766-71.

  Low-density lipoproteins (LDLs) represent the most important treatable risk factors for coronary artery disease. Although it has been previously shown that hypercholesterolemia stimulates theLow-density lipoproteins (LDLs) represent the most important treatable risk factors for coronary artery disease. Although it has been previously shown that hypercholesterolemia stimulates the endothelin system, the effects of increased levels of LDL on endothelial endothelin receptors have not been previously studied. In particular, the influence of native and oxidatively modified LDLs (nLDLs and oxLDLs) and the regulatory mechanisms in endothelial cells are currently unknown. Human endothelial cells almost exclusively express the endothelin receptor type B (ET(B)). Therefore, the effect of nLDL and oxLDL on the expression of ET(B) was studied in primary cultures of human umbilical vein endothelial cells (HUVEC). HUVEC were stimulated by nLDL and oxLDL in a time-dependent (1-12 hrs) and dose-dependent (25-100 microg/ml) manner. To analyze signal transduction pathways involved in the regulation of ET(B), protein kinase C (PKC) was inhibited using 100 nM Ro-31-8220. The mRNA expression of ET(B) was determined by quantitative reverse transcription-polymerase chain reaction and ET(B) protein expression by Western blot. Native LDL induced ET(B) mRNA after 1 hr (100 microg/ml, 199 +/- 35%, n = 15, P < 0.05 vs. control). Stimulation of HUVEC with oxLDL increased ET(B) mRNA expression (1 hr, 100 microg/ml oxLDL: 308 +/- 48%, n = 15, P < 0.05 vs. control) as well. Induction of ET(B) was also found on the protein level. nLDL was even more potent than oxLDL in inducing ET(B) protein expression. Induction of ET(B) expression by oxLDL is mediated by PKC. These data demonstrate that low-density lipoproteins even independent of oxidative modification are potent inducers of ET(B) receptors at the mRNA and protein level in HUVEC. Given the nitric oxide-releasing capacity of endothelial ET(B) receptors, this effect may represent a possible vasoprotective mechanism.

• Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cells.

  Authors: Claudia Stielow, Rusan A Catar, Gregor Muller, Kirstin Wingler, Peter Scheurer, Harald H H W Schmidt, Henning Morawietz

  Biochemical and biophysical research communications. 05/2006; 344(1):200-5.

  In this study, we investigated effects of a novel NAD(P)H oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870) on oxidized low-density lipoproteinIn this study, we investigated effects of a novel NAD(P)H oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870) on oxidized low-density lipoprotein (oxLDL)-mediated reactive oxygen species (ROS) formation in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were cultured to confluence and ROS formation was induced with 50microg/ml oxLDL for 2h. ROS formation was detected by chemiluminescence (CL) using the Diogenes reagent. OxLDL induced ROS formation in human endothelial cells (171+/-12%; n=10, P<0.05 vs. control). This augmented ROS formation in response to oxLDL was completely inhibited by the Nox inhibitor VAS2870 (101+/-9%; n=7, P<0.05 vs. oxLDL). Similar results were obtained with superoxide dismutase (91+/-7%; n=7, P<0.05 vs. oxLDL). However, the Nox4 mRNA expression level was neither changed by oxLDL nor VAS2870. We conclude that VAS2870 could provide a novel strategy to inhibit the augmented endothelial superoxide anion formation in response to cardiovascular risk factors.

• Native and oxidized low-density lipoproteins stimulate endothelin-converting enzyme-1 expression in human endothelial cells.

  Authors: Bernd Niemann, Susanne Rohrbach, Rusan A Catar, Gregor Muller, Matthias Barton, Henning Morawietz

  Biochemical and biophysical research communications. 10/2005; 334(3):747-53.

  This study addressed the question how different lipoproteins modulate the expression of endothelin-converting enzyme-1 (ECE-1) in human endothelial cells. The effect of native and oxidizedThis study addressed the question how different lipoproteins modulate the expression of endothelin-converting enzyme-1 (ECE-1) in human endothelial cells. The effect of native and oxidized low-density lipoproteins (nLDL, oxLDL) on expression of ECE-1, prepro-endothelin-1, and endothelin-1 peptide was studied in primary cultures of human endothelial cells. Native and oxidized LDL increased ECE-1 mRNA after 1 h, reaching its maximum at 100 microg/ml (1.9- and 2.5-fold, respectively). Furthermore, ECE-1 protein expression, prepro-endothelin-1 mRNA, and endothelin-1 peptide release were increased in response to nLDL or oxLDL. Induction of ECE-1 by nLDL and of prepro-endothelin-1 by oxLDL was reduced by protein kinase C inhibition. Increased expression of ECE-1 mRNA by oxLDL and of prepro-endothelin-1 by nLDL was blocked by an angiotensin II receptor type 1 antagonist. Our data provide evidence for a new mechanism how increased LDL plasma levels might contribute to enhanced endothelin-1 release in patients with hypercholesterolemia.

• Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

  Authors: Claudia Goettsch, Winfried Goettsch, Gregor Muller, Jochen Seebach, Hans-Joachim Schnittler, Henning Morawietz

  Biochemical and Biophysical Research Communications.

  Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCRNicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.