First 11 pages of thesis. - OPUS - Universität Würzburg

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vessels with the iNOS specific inhibitor 1400W significantly reduced superoxide levels, indicating that iNOS itself contributes to superoxide production in atherosclerosis. To the best of our knowledge this is the first evidence supporting superoxide generation by iNOS in atherosclerosis. In contrast, ample evidence supports eNOS “uncoupling” and subsequent superoxide, instead of nitric oxide production 151, 206 . The mechanism of superoxide generation by iNOS differs from eNOS and nNOS. The latter two enzymes produce superoxide from the heme centers of their oxygenase domains 35, 117 , while iNOS produces superoxide from the reductase domain 136 . While 100 µM of L-arginine was sufficient to block superoxide generation from nNOS 117 , there was no effect of 100 µM L-arginine on iNOS mediated superoxide generation 136 . Higher concentrations of L-arginine (1mM–5mM) only reduced, but did not abolish superoxide production, indicating that superoxide generation by iNOS may even occur when substrate is not limited 136 . Xia et al. reported that induction of iNOS in macrophages reduced L-arginine concentrations, indicating high substrate demand 37 . These authors hypothesized that iNOS may catalyze nitric oxide from the oxygenase- and superoxide from the reductase domain simultaneously, which could directly lead to peroxynitrite formation. Quantification of tissue 3-nitrotyrosine, considered a footprint of peroxynitrite mediated protein damage showed reduced levels of 3-nitrotyrosine in apoE/iNOS dko vessels, compared to apoE ko vessels. Peroxynitrite oxidizes BH4, a cofactor of NOS 207 which in its oxidized form causes uncoupling of the constitutive NOS enzymes 103 . Further, peroxynitrite can directly cause irreversible uncoupling and inactivation of nNOS 208 . iNOS mediated peroxynitrite formation and its high substrate demand may thus cause - 74 -
uncoupling of constitutive NOS isoforms, resulting in further superoxide production. The residual peroxynitrite detected in apoE/iNOS dko might be due to the reaction of nitric oxide from constitutive NOS with superoxide generated from myeloperoxidase, NADPH oxidase and xanthine oxidase. iNOS and NADPH oxidase are pro inflammatory enzymes and known to be expressed in atherosclerotic vessels 43, 164 and the expression of both enzymes coincides with localized oxidative stress in the lesions. NADPH oxidase is proposed to negatively regulate the expression of iNOS in smooth muscle cells 209 . While increased nitric oxide generated by iNOS is suppresses the expression and activity of NADPH oxidase under certain inflammatory conditions 210 interleukin-1 induced nitric oxide (i.e iNOS) increases NADPH oxidase activity in smooth muscle cells of coronary arteries 211 . Since complex interactions exists between iNOS and NADPH oxidase we determined the contribution of NADPH oxidase to superoxide production in apoE ko and apoE/iNOS dko atherosclerosis. Consistent with the previous observation that NADPH oxidase is one of the major sources of superoxide generation in atherosclerosis 44 , we found that NADPH oxidase significantly contributes to superoxide production in atherosclerosis. The reduction of superoxide production mediated by the NADPH oxidase inhibitor apocynin was similar to that observed in apoE/iNOS dko animals and inhibition of NADPH oxidase by apocynin did not further reduce the superoxide levels in apoE/iNOS dko vessels. These results suggest that the decrease of superoxide production observed in apoE/iNOS dko aorta is largely due to decreased NADPH oxidase activity in this genotype. - 75 -
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Insight into oxidative stress media
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Eidesstattliche Erklärungen Hiermi
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Acknowledgements First and foremost
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“Research is to see what everybod
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Chapter 2 37 2.0 Materials and Meth
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Summary and Hypothesis 76 Zusammenf
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1.0 Introduction Atherosclerosis, t
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which contribute to the lipid rich
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1.2 Risk Factors Atherosclerosis ha
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1.2.2 Environmental factors 1. High
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1.3 Oxidative Stress Oxidative stre
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peroxynitrite 38 which by itself ca
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Pharmacological inhibition of nitri
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1.4 The nitric oxide pathway in ath
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of NADPH, FAD and FMN, whereas the
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isoforms to the regulatory and targ
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perinuclear/golgi region within the
Page 36 and 37: nNOS interacts with several protein
Page 38 and 39: iNOS expression is regulated transc
Page 40 and 41: transport pathway and is an essenti
Page 42 and 43: Interestingly, BH4 supplementation
Page 44 and 45: the survival rate, as apoE/nNOS dko
Page 46 and 47: The seemingly opposing effects of i
Page 48 and 49: The purpose of the study presented
Page 50 and 51: 2.0 Materials and Methods 2.1 Mater
Page 52 and 53: Reagents Source For Immunohistochem
Page 54 and 55: Others Reagents Source Pegulated Su
Page 56 and 57: 2.2 Methods 2.2.1 Detection of free
Page 58 and 59: 2.2.1.2 Principle of ESR ESR spectr
Page 60 and 61: spectra 190 . Addition of exogenous
Page 62 and 63: ings were prepared as mentioned abo
Page 64 and 65: X-band EMX spectroscope (Bruker Bio
Page 66 and 67: minutes. At last sections were wash
Page 68 and 69: 3.0 Results 3.1 eNOS is a significa
Page 70 and 71: 3.2 eNOS deletion decreases vascula
Page 72 and 73: 3.3 nNOS contributes little to vasc
Page 74 and 75: 3.5 iNOS contributes significantly
Page 76 and 77: Figure 13. Determination of iNOS de
Page 78 and 79: 3.8 iNOS deletion influences NADPH
Page 80 and 81: 4.0 Discussion 4.1 eNOS is a major
Page 82 and 83: in our apoE/eNOS dko model, these a
Page 84 and 85: under conditions of substrate or co
Page 88 and 89: Summary and Hypothesis The principl
Page 90 and 91: Zusammenfassung und Hypothese Stick
Page 92 and 93: Bibliography 1. Gimbrone MA, Jr. Va
Page 94 and 95: xanthine oxidase contributes to vas
Page 96 and 97: expression of adhesion molecules an
Page 98 and 99: transforming growth factor-beta 1.
Page 100 and 101: generation from neuronal nitric oxi
Page 102 and 103: 148. Lee PC, Wang ZL, Qian S, Watki
Page 104 and 105: 174. Mayr U, Zou Y, Zhang Z, Dietri
Page 106 and 107: implications in intracellular fluor
Page 108 and 109: Definitions Thesaurus • Allograft
Page 110 and 111: • Claudication refers to leg pain
Page 112 and 113: cases per year in the US. It is a n
Page 114 and 115: Curriculum Curriculum vitae vitae N
Page 116 and 117: presentation at the 37 th Annual co
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First 11 pages of thesis. - OPUS - Universität Würzburg

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