Studies on Experimental Models

Oxidative Stress in Applied Basic Research and Clinical Practice

Editor-in-Chief Donald Armstrong

For other titles published in this series, go to www.springer.com/series/8145

Note from the Editor-in-Chief All books in this series illustrate point-of-care testing and critically evaluate the potential of antioxidant supplementation in various medical disorders associated with oxidative stress. Future volumes will be updated as warranted by emerging new technology, or from studies reporting clinical trials. Donald Armstrong Editor-in-Chief

Samar Basu  •  Lars Wiklund Editors

Studies on Experimental Models

Editors Samar Basu Professor of Biochemistry and Medical Inflammation Director of Chaire d’Excellence Laboratorie de Biochimie Biologie Moléculaire et Nutrition Faculté de Pharmacie Université d’Auvergne 28 Place Henri-Dunant BP 38 63001 Clermont-Ferrand France and Head, Oxidative stress and Inflammation Department of Public Health and Caring Sciences Faculty of Medicine Uppsala University SE-751 85 Uppsala Sweden [email protected] [email protected]

Lars Wiklund Professor of Anesthesiology and Intensive Care Medicine Department of Surgical Sciences/ Anesthesiology and Intensive Care Medicine Faculty of Medicine Uppsala University SE-751 85 Uppsala Sweden [email protected]

ISBN 978-1-60761-955-0 e-ISBN 978-1-60761-956-7 DOI 10.1007/978-1-60761-956-7 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011923976 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written ­permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface

The notion that reactive oxygen species are involved in diseases originated in the 1930s when scientists realized that radiation injury may lead to formation of free radicals and in turn initiate cancer and other pathologies. Even though research in free radicals has been stretched exponentially in the last three decades, there are still major questions as to what extent free radicals are involved in health and various diseases. In recent years, there is a growing recognition that free radicals, and thereby oxidative stress, is involved in atherosclerosis, cardiovascular diseases, cancer, ischemia–reperfusion injury, radiation injury and neurological diseases, etc. In addition, free radicals are perhaps involved in both aging and redox process. Despite the great amount of research being carried out in the field, there are still uncertainties about the overall mechanisms behind free radical-related pathologies, as well as how free radicals may play a fundamental role in protecting cells through redox signaling. With these concepts in mind, there is a wide-spread consensus today that the use of antioxidants as a therapeutic approach may counteract free radical-mediated pathologies. However, the role of antioxidants in normal physiology and redox signaling is still in its infancy. Since oxidative stress is related to various diseases and pathologies, scientists are eager to study the disease in humans. However, it is not always ethical to study all the aspects of the disease in humans; consequently, it is mandatory to study the disease process and the mechanisms behind it through experimental models. The models generally involve animals, in  vitro/cell culture studies, and even in primates and humans to a certain extent. This book contains mainly data on the experimental models or review of such models of oxidative stress in various diseases, and is divided into six parts that present a sketch of models in humans, animals and in vitro methods. Part I deals with diabetes, which includes the role of oxidative stress and antioxidant therapies in experimental models of diabetes, diabetes complications, micronutrient intake and its relevance to atherosclerosis and insulin resistance. Part II deals with stroke, including arachidonic acid metabolism and the role of alpha-tocotrienol as a therapeutic agent, assessment of oxidative stress, heat-shock proteins and doxorubicininduced oxidative stress in the heart, as well as biomarkers of oxidative stress in cardiovascular diseases. Part III deals with neurology, which includes MPTP and

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oxidative stress, oxidative stress in Alzheimer’s disease, retinal disturbances in patients and animals models with Huntington’s, Parkinson’s and Alzheimer’s disease, stress gene regulation in Alzheimer’s blood cells, Gpx4 knockout mice and transgenic mice in aging, experimental models of myocardial, and cerebral ischemia. Part IV deals with ocular diseases, including neovascular models of the rabbit eye and purinergic signaling in volume regulation of glial cells in rat retina. Part V is concerned with toxicological/environmental aspects that contain Helicobacter Pylori-induced oxidative stress and inflammation, experimental models for ionizing radiation research, cigarette smoke-induced oxidative stress in preclinical models, smokers and patients with airways disease, exhaled breath condensate biomarkers in airway inflammation in COPD, induction of oxidative stress by iron/ascorbate in isolated mitochondria and by UV irradiation in human skin, carbon tetrachlorideinduced oxidative stress, lipid metabolites after exposure of toxicants, oxidative stress in porcine endotoxemia and exercise as a model to study oxidative stress. The final part concerns in vitro/cell culture models that contain mitochondrial oxidative stress, protection against oxidant-induced neuronal cell injury, oxidative DNA biomarkers, oxidative stress in cell culture, animals and humans, role of cAMP and G protein signaling in cardiovascular dysfunction, cellular and chemical assays of antioxidants, and arsenic-induced oxidative stress. We hope that this book will contribute to the advancement of oxidative stress research using appropriate animal models and will serve as a valuable reference for basic and clinical scientists. Editor Co-Editor

Samar Basu Lars Wiklund

Contents

Part I  Diabetes Role of Oxidative Stress and Targeted Antioxidant Therapies in Experimental Models of Diabetic Complications..................................... Judy B. de Haan, Karin A. Jandeleit-Dahm, and Terri J. Allen

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Experimental Models of Oxidative Stress Related to Cardiovascular Diseases and Diabetes...................................................... Maria D. Mesa, Concepcion M. Aguilera, and Angel Gil

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Part II  Cardiovascular Arachidonic Acid Metabolism and Lipid Peroxidation in Stroke: Alpha-Tocotrienol as a Unique Therapeutic Agent...................................... Cameron Rink, Savita Khanna, and Chandan K. Sen Assessment of Oxidative Stress in the Brain of Spontaneously Hypertensive Rat and Stroke-Prone Spontaneously Hypertensive Rat Using by Electron Spin Resonance Spectroscopy.................................. Fumihiko Yoshino, Kyo Kobayashi, and Masaichi-Chang-il Lee

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Small Heat Shock Proteins and Doxorubicin-Induced Oxidative Stress in the Heart.......................................................................... 105 Karthikeyan Krishnamurthy, Ragu Kanagasabai, Lawrence J. Druhan, and Govindasamy Ilangovan Oxidative Stress in Cardiovascular Disease: Potential Biomarkers and Their Measurements........................................... 131 Subhendu Mukherjee and Dipak K. Das In vivo Imaging of Antioxidant Effects on NF-k B Activity in Reporter Mice................................................................................ 157 Ingvild Paur, Harald Carlsen, and Rune Blomhoff vii

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Part III  Neurology MPTP and Oxidative Stress: It’s Complicated!............................................ 187 V. Jackson-Lewis, M.A. Tocilescu, R. DeVries, D.M. Alessi, and S. Przedborski Oxidative Stress in Alzheimer’s Disease: A Critical Appraisal of the Causes and the Consequences.......................... 211 Jaewon Chang, Sandra Siedlak, Paula Moreira, Akihiko Nunomura, Rudy J. Castellani, Mark A. Smith, Xiongwei Zhu, George Perry, and Gemma Casadesus Retinal Disturbances in Patients and Animal Models with Huntington’s, Parkinson’s and Alzheimer’s Disease............................ 221 C. Santano, M. Pérez de Lara, and J. Pintor Stress Gene Deregulation in Alzheimer Peripheral Blood Mononuclear Cells........................................................................................... 251 Olivier C. Maes, Howard M. Chertkow, Eugenia Wang, and Hyman M. Schipper The Use of Gpx4 Knockout Mice and Transgenic Mice to Study the Roles of Lipid Peroxidation in Diseases and Aging................................ 265 Qitao Ran and Hanyu Liang An Experimental Model of Myocardial and Cerebral Global Ischemia and Reperfusion............................................................................... 279 Lars Wiklund and Samar Basu Part IV  Ocular Diseases Neovascular Models of the Rabbit Eye Induced By Hydroperoxide........... 303 Toshihiko Ueda, Takako Nakanishi, Kazushi Tamai, Shinichi Iwai, and Donald Armstrong Purinergic Signaling Involved in the Volume Regulation of Glial Cells in the Rat Retina: Alteration in Experimental Diabetes..................... 319 Andreas Bringmann Part V  Toxicology/Environmental Helicobacter pylori-Induced Oxidative Stress and Inflammation................ 343 Hyeyoung Kim and Young-Joon Surh

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Experimental Models for Ionizing Radiation Research............................... 371 Kristin Fabre, William DeGraff, John A. Cook, Murali C. Krishna, and James B. Mitchell Experimental Models to Study Cigarette Smoke-Induced Oxidative Stress In Vitro and In Vivo in Preclinical Models, and in Smokers and Patients with Airways Disease................................................................. 399 Hongwei Yao and Irfan Rahman Exhaled Breath Condensate Biomarkers of Airway Inflammation and Oxidative Stress in COPD........................................................................ 421 Paolo Montuschi Induction of Oxidative Stress by Iron/Ascorbate in Isolated Mitochondria and by UV Irradiation in Human Skin................................. 441 Ingrid Wiswedel, Wolfgang Augustin, Sven Quist, Harald Gollnick, and Andreas Gardemann Carbon Tetrachloride-Induced Hepatotoxicity: A Classic Model of Lipid Peroxidation and Oxidative Stress...................... 467 Samar Basu Enhanced Urinary Excretion of Lipid Metabolites Following Exposure to Structurally Diverse Toxicants: A Unique Experimental Model for the Assessment of Oxidative Stress............................................... 481 Francis C. Lau, Manashi Bagchi, Shirley Zafra-Stone, and Debasis Bagchi Oxidative Stress in Animal Models with Special Reference to Experimental Porcine Endotoxemia.......................................................... 497 Miklós Lipcsey, Mats Eriksson, and Samar Basu Models and Approaches for the Study of Reactive Oxygen Species Generation and Activities in Contracting Skeletal Muscle.......................... 511 Malcolm J. Jackson Exercise as a Model to Study Interactions Between Oxidative Stress and Inflammation.................................................................................. 521 Christina Yfanti, Søren Nielsen, Camilla Scheele, and Bente Klarlund Pedersen Exercise as a Model to Study Oxidative Stress.............................................. 531 Mari Carmen Gomez-Cabrera, Fabian Sanchis-Gomar, Vladimir Essau Martinez-Bello, Sandra Ibanez-Sania, Ana Lucia Nascimento, Li Li Ji, and Jose Vina

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Part VI  In Vitro/Tissue Culture Models of Mitochondrial Oxidative Stress.................................................... 545 Enrique Cadenas and Alberto Boveris Protection of Oxidant-Induced Neuronal Cells Injury by a Unique Cruciferous Nutraceutical............................................................................... 563 Zhenquan Jia, Soumya Saha, Hong Zhu, Yunbo Li, and Hara P. Misra Oxidatively Generated Damage to DNA and Biomarkers........................... 579 Jean Cadet, Thierry Douki, and Jean-Luc Ravanat Measuring Oxidative Stress in Cell Cultures, Animals and Humans: Analysis and Validation of Oxidatively Damaged DNA............................... 605 Hanna L. Karlsson and Lennart Möller The Roles of cAMP and G Protein Signaling in Oxidative Stress-Induced Cardiovascular Dysfunction................................................. 621 Soumya Saha, Zhenquan Jia, Dongmin Liu, and Hara P. Misra Cellular and Chemical Assays for Discovery of Novel Antioxidants in Marine Organisms....................................................................................... 637 Tim Hofer, Tonje Engevik Eriksen, Espen Hansen, Ingrid Varmedal, Ida-Johanne Jensen, Jeanette Hammer-Andersen, and Ragnar Ludvig Olsen Arsenic-Induced Oxidative Stress: Evidence on In Vitro Models of Cardiovascular, Diabetes Mellitus Type 2 and Neurodegenerative Disorders........................................................................................................... 659 Rubén Ruíz-Ramos, Patricia Ostrosky-Wegman, and Mariano E. Cebrián Index . ............................................................................................................... 681

Contributors

Concepcion M. Aguilera Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Centre of Biomedical Research, University of Granada, Campus de la Salud, Armilla, Granada, Spain Terri J. Allen Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia D.M. Alessi Graduate student, Integrated Program of Cellular, Molecular and Biophysical studies at Columbia University, USA Donald Armstrong Department of Ophthalmology, University of Florida College of Medicine, Gainesville, Florida, USA Wolfgang Augustin Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany Debasis Bagchi InterHealth Research Center, Benicia, CA, USA; Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA Manashi Bagchi InterHealth Research Center, Benicia, CA, USA Samar Basu Laboratorie de Biochimie, Biologie Moléculaire et Nutrition Faculté de Pharmacie, Université d’Auvergne 28, Clermont-Ferrand, France; Oxidative stress and Inflammation, Department of Public Health and Caring Sciences, Faculty of Medicine, Uppsala University, Uppsala, Sweden

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Contributors

Rune Blomhoff Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Alberto Boveris Physical Chemistry, School of Pharmacy & Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Andreas Bringmann Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany Enrique Cadenas Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, USA Jean Cadet Laboratoire “Lésions des Acides Nucléiques”, SCIB-UMR-E n°3 (CEA/UJF), Institut Nanosciences et Cryogénie, CEA/Grenoble, 38054 Grenoble Cedex 9, France Harald Carlsen Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Gemma Casadesus Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA Rudy J. Castellani Department of Pathology, University of Maryland, Baltimore, MD, USA Mariano E. Cebrián Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Ave. Instituto Politecnico National, Mexico, DF, Mexico Jaewon Chang Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA Howard M. Chertkow Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada H3T 1E2; Departments of Neurology, Neurosurgery and Medicine (Geriatrics), McGill University, Montréal, Québec, Canada H3G 146 John A. Cook Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

Contributors

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Dipak K. Das Cardiovascular Research Center, University of Connecticut Health Center, School of Medicine, Farmington, CT, USA William DeGraff Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Judy B. de Haan Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia R. DeVries Departments of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Thierry Douki Laboratoire “Lésions des Acides Nucléiques”, SCIB-UMR-E n°3 (CEA/UJF), Institut Nanosciences et Cryogénie, CEA/Grenoble, 38054 Grenoble Cedex 9, France Lawrence J. Druhan Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA Tonje Engevik Eriksen MabCent-SFI, University of Tromsø, Tromsø, Norway Mats Eriksson Department of Anaesthesia and Intensive Care, Uppsala University, Uppsala, Sweden Kristin Fabre Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Andreas Gardemann Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany Angel Gil Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Centre of Biomedical Research, University of Granada, Campus de la Salud, Armilla, Granada, Spain Harald Gollnick Department of Dermatology and Venereology, Otto-von-Guericke University, Magdeburg, Germany Mari Carmen Gomez-Cabrera Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain

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Contributors

Jeanette Hammer-Andersen MabCent-SFI, University of Tromsø, Tromsø, Norway Espen Hansen MabCent-SFI, University of Tromsø, Tromsø, Norway Tim Hofer MabCent-SFI, University of Tromsø, Tromsø, Norway Sandra Ibanez-Sania Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain Govindasamy Ilangovan Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA Shinichi Iwai Departments of Pharmacology and Ophthalmology, Showa University School of Medicine, Tokyo, Japan Malcolm J. Jackson School of Clinical Sciences, University of Liverpool, Liverpool, UK V. Jackson-Lewis Departments of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Karin A. Jandeleit-Dahm Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia Ida-Johanne Jensen MabCent-SFI, University of Tromsø, Tromsø, Norway Li Li Ji The Biodynamics Laboratory, Department of Kinesiology, University of Wisconsin at Madison, WI, USA Zhenquan Jia Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA Ragu Kanagasabai Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA Hanna L. Karlsson Unit for Analytical Toxicology, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden

Contributors

Savita Khanna Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA Hyeyoung Kim Department of Food and Nutrition, Brain Korea 21 Project, College of Human Ecology, Yonsei University, Seoul, South Korea Kyo Kobayashi Department of Clinical Care Medicine, Division of Pharmacology, Kanagawa Dental College, Yokosuka, Japan Murali C. Krishna Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Karthikeyan Krishnamurthy Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA Francis C. Lau InterHealth Research Center, Benicia, CA, USA Masaichi-Chang-il Lee Department of Clinical Care Medicine, Division of Pharmacology, Kanagawa Dental College, Yokosuka, Japan Yunbo Li Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA Hanyu Liang Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Miklós Lipcsey Department of Anaesthesia and Intensive Care, Uppsala University Hospital, Uppsala, Sweden Dongmin Liu Department of Human Food, Nutrition and Exercise, Virginia Tech, Blacksburg, VA, USA Olivier C. Maes Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada Vladimir Essau Martinez-Bello Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain

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Contributors

Maria D. Mesa Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Centre of Biomedical Research, University of Granada, Campus de la Salud, Armilla, Granada, Spain Hara P. Misra Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA James B. Mitchell Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Lennart Möller Unit for Analytical Toxicology, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden Paolo Montuschi Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy Paula Moreira Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, Coimbra, Portugal Subhendu Mukherjee Cardiovascular Research Center, University of Connecticut Health Center, School of Medicine, Farmington, CT, USA Takako Nakanishi Departments of Ophthalmology, Showa University School of Medicine, Tokyo, Japan Ana Lucia Nascimento Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain S. Nielsen The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen Faculty of Health Sciences, Copenhagen, Denmark Akihiko Nunomura Department of Neuropsychiatry, University of Yamanashi, Yamanashi, Japan Ragnar Ludvig Olsen MabCent-SFI, University of Tromsø, Tromsø, Norway Patricia Ostrosky-Wegman Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Circuito Escolar, Cd. Universitaria, Mexico, DF, Mexico

Contributors

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Ingvild Paur Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Bente K. Pedersen The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark George Perry Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA; Department of Biology, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, Coimbra, Portugal; UTSA Neurosciences Institute, University of Texas at San Antonio, San Antonio, TX, USA M. Pérez de Lara Departamento de Bioquímica y Biología Molecular, Escuela de Optica UCM, Madrid, Spain J. Pintor Departamento de Bioquímica y Biología Molecular, Escuela de Optica UCM, Madrid, Spain S. Przedborski Departments of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Sven Quist Department of Dermatology and Venereology, Otto-von-Guericke University, Magdeburg, Germany Irfan Rahman Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, NY, USA Qitao Ran Department of Cellular and Structural Biology; Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio; South Texas Veterans Health Care System, San Antonio, TX, USA; Jean-Luc Ravanat Laboratoire “Lésions des Acides Nucléiques,” SCIB-UMR-E n°3 (CEA/UJF), Institut Nanosciences et Cryogénie, CEA/Grenoble, 38054 Grenoble Cedex 9, France Cameron Rink Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA

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Contributors

Rubén Ruíz-Ramos Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Mexico, DF, Mexico Soumya Saha Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA; Department of Human Food, Nutrition and Exercise, Virginia Tech, Blacksburg, VA, USA C. Santano Departamento de Bioquímica y Biología Molecular, Escuela de Optica UCM, Madrid, Spain Fabian Sanchis-Gomar Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain C. Scheele The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark Hyman M. Schipper Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada; Departments of Neurology, Neurosurgery and Medicine (Geriatrics), McGill University, Montréal, Québec, Canada Chandan K. Sen Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA Sandra Siedlak Department of Pathology, Case Western Reserve University, Cleveland, OH, USA Mark A. Smith Department of Pathology, Case Western Reserve University, Cleveland, OH, USA Young-Joon Surh Department of Molecular Medicine and Biopharmaceutics, Graduate School of Convergence Sciences and Technology, Seoul National University, Seoul, South Korea Kazushi Tamai Department of Ophthalmology, Nagoya City University School of Medicine, Nagoya, Japan M.A. Tocilescu Department of Neurology, Center for Motor Neuron Biology and Disease, Departments of Pathology and Cell Biology, Columbia University, New York, NY, USA

Contributors

Toshihiko Ueda Department of Ophthalmology, Showa University School of Medicine, Tokyo, Japan Ingrid Varmedal MabCent-SFI, University of Tromsø, Tromsø, Norway Jose Vina Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain C. Vives-Bauza Department of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Eugenia Wang Gheens Center on Aging, and Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, KY, USA Lars Wiklund Department of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, 751 85 Uppsala, Sweden Ingrid Wiswedel Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany Hongwei Yao Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, NY, USA Christina Yfanti The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark Fumihiko Yoshino Department of Clinical Care Medicine, Division of Pharmacology, Kanagawa Dental College, Yokosuka, Japan Shirley Zafra-Stone InterHealth Research Center, Benicia, CA, USA Hong Zhu Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA Xiongwei Zhu Department of Pathology, Case Western Reserve University, Cleveland, OH, USA

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Part I

Diabetes

Role of Oxidative Stress and Targeted Antioxidant Therapies in Experimental Models of Diabetic Complications Judy B. de Haan, Karin A. Jandeleit-Dahm, and Terri J. Allen

Abstract  Diabetic patients, whether of type 1 or type 2 origin, are at greater risk of developing complications of the vasculature than non-diabetic patients. Macrovascular complications such as diabetes-associated atherosclerosis lead to accelerated and often more advanced lesions than seen in the general population. Microvascular complications such as nephropathy, retinopathy and neuropathy as well as diabetic cardiomyopathy are further complications associated with the diabetic milieu. Understanding the mechanisms leading to and accelerating these complications is a major research initiative of many laboratories. To facilitate these studies, the design and use of appropriate animal models has been central to the study of these diabetic complications. A new and emerging concept underpinning many of these end-organ complications is oxidative stress, particularly of mitochondrial origin, which is understood to play a critical role in the initiation and progression of these diabetic complications. Thus the development of experimental models that specifically delineate the cause and role of ROS in diabetic complications is now becoming a major research area. This chapter focuses on some of the latest oxidative stress-driven experimental models of diabetic complications. Use of the ApoE/GPx1 double-knockout mouse has revealed the importance of antioxidant defense in limiting accelerated diabetes-associated atherosclerosis and diabetic nephropathy, while RAGE knockout mice have shown that oxidative stress is inextricably linked with pathophysiological cell signaling, particularly through RAGE. The use of NOX knockout mice is shedding light on the contribution of the NADPH oxidases to the ROS milieu as well as the contribution of the various isoforms (NOX 1, 2 and 4) to the individual diabetic complications. Furthermore, these models are helping to understand the types of ROS involved and their cellular location, which may help in the specific targeting of these ROS to reduce ROSmediated pathogenesis. For example, antioxidants that target mitochondrial ROS (location) or ROS such as hydrogen peroxide (specificity) may offer an alternate T.J. Allen (*) Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, St Kilda Road Central, Melbourne, VIC 8008, Australia e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_1, © Springer Science+Business Media, LLC 2011

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approach to reduce diabetes-driven oxidative stress. It is only via manipulation of experimental models of diabetes-driven oxidative stress that the contribution of the various ROS will be revealed, and only then that effective treatment regimens can be designed to lessen the effect of oxidative stress on diabetic complications. Keywords  Antioxidant defense • Diabetic complications • Ebselen • Experimental models • Glutathione peroxidase • Oxidative stress

1 Introduction Diabetes mellitus is a metabolic disorder that is characterized by chronic hyperglycemia with disturbances in carbohydrate, fat and protein metabolism and occurs as a result of defects in insulin secretion and/or action [1]. Whether diabetes occurs as a result of type 1, the early-onset and predominantly insulin-dependent form, or type 2, the late-onset form that is associated with metabolic syndrome, obesity and insulin-resistance, individuals with diabetes are at greater risk of developing diabetes-associated complications [2, 3]. The predominant complications include cardiovascular disease, nephropathy, retinopathy, neuropathy and a specific impairment in the heart muscle leading to cardiomyopathy [4]. It is now well accepted that most diabetic complications arise from chronic hyperglycemic damage to the vascular system [5]. Vascular disease can be separated into that affecting the macrovasculature resulting in atherosclerosis of major vessels and/or stroke, and microvascular complications resulting in retinopathy, nephropathy and neuropathy. Diabetic cardiomyopathy is understood to occur as a result of abnormal myocardial metabolism in diabetes, rather than as a result of micro- or macro-vascular disease [6–8]. While many studies address the causative mechanisms of the individual ­diabetes-driven complication, it is now becoming increasingly apparent that oxidative stress is an important underpinning phenomenon that assists with the progression towards more severe and often fatal complications. Evidence from numerous studies suggests an important causal role for increased reactive oxygen species (ROS), particularly mitochondrial ROS, in the pathogenesis of the major complications associated with diabetes [9]. Several pathways, including the polyol pathway [10], increases in advanced glycation end products (AGEs) [11], activation of ­protein kinase C [12] and increases in hexosamine flux, have been identified where hyperglycemia triggers increased ROS production that in turn may initiate, progress or amplify end-organ damage in diabetes. One postulate suggests that all of these pathways are activated as a result of glucose-induced overproduction of superoxide by the mitochondrial electron transport chain [2]. Indeed it has been estimated that 1–2% of all electrons passing through the respiratory chain contribute to the ­formation of superoxide [13, 14], with the rate of production varying greatly depending on the environment and/or disease state [14]. Other potential sources of ROS production include nitric oxide synthases (NOS), nicotinamide adenine

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dinucleotide phosphate (NADPH) oxidases (NOX), xanthine oxidase, lipoxygenase and cytochrome P450 mono-oxygenases [13, 15]. Particular attention has focused on the members of the NOX/DUOX family of NADPH oxidases since these enzymes mediate physiological functions such as host defense, cell signaling, and thyroid hormone biosynthesis through the generation of ROS, including superoxide anion and hydrogen peroxide [16]. However, it is becoming increasingly apparent that alterations in diabetes-driven cellular ROS arise not only as a consequence of overproduction of ROS, but also as a result of ineffective removal by antioxidant defenses [17, 18]. The design and use of appropriate animal models to study diabetic complications has been examined in several reviews covering mouse models of type 1 and type 2 diabetes as well as related phenotypes such as obesity and insulin resistance [5, 19–22]. However, the development of experimental models that specifically delineate the cause and role of ROS in diabetic complications is now becoming a major research area, enabling both an understanding of the mechanisms of ROSmediated damage as well as facilitating the design of targeted therapeutic approaches to limit diabetes-mediated ROS action. This chapter will focus on some of the latest oxidative stress-induced experimental models of diabetic complications with particular emphasis on cardiovascular disease, nephropathy, and diabetic cardiomyopathy.

2 Diabetes-Associated Atherosclerosis Diabetes mellitus is a major pro-atherosclerosis risk factor, with a two- to fourfold higher incidence of cardiovascular disease in diabetic patients than in the general population [23]. Other risk factors include hyperglycemia, dyslipidemia, hypertension and obesity. However, these risk factors only partly explain the more advanced lesions [24] and increased incidence of cardiovascular disease observed in these patients. Understanding the underlying mechanisms that accelerate diabetes-­ associated atherosclerosis remains an important research area and various pathways have been implicated that include the biochemical process of advanced glycation [25] and the receptor for AGEs, RAGE [26]. In addition, various proteins that have been implicated in the atherosclerotic process per se have been shown to be upregulated in the diabetic condition. These include vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CTGF) [27]. Strong evidence now suggests that ROS derived from the hyperglycemia-driven increase in mitochondrial electron transport chain activity [9], glucose autoxidation [28] and enzymes such as NAD(P)H oxidase play a causal role in mediating many of the pro-atherogenic changes observed [29]. Indeed, ROS are known to upregulate a number of pro-atherogenic processes such as monocyte infiltration, platelet activation [30], smooth muscle cell migration [31], cell adhesion [32], release of CTGF [33] and increased production of AGEs [28]. Specifically, the accumulation

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of oxidized low-density lipoproteins (oxLDL) in vessel walls is an early initiator of atherosclerotic events [34]. In turn, oxLDL is atherogenic for several reasons, namely (1) it is chemotactic for circulating monocytes, the cellular precursors of arterial macrophages, (2) it is responsible for inhibiting the migration of ­macrophages from the aortic wall, thus trapping them inside the lesion and (3) it is directly cytotoxic to endothelial cells, thus aiding in the erosion of the endothelial surface and promoting thrombosis [35–37]. Furthermore, macrophages preferentially take up oxLDL, thereby promoting atherosclerosis [38], while the oxidative bursts of macrophages and neutrophils recruited to inflammatory sites further ­compound the oxidative insult [37]. Importantly, a heightened state of oxidative stress has been observed in diabetic patients [39]. Several laboratories, including ours, have focused on the role of antioxidant defense in regulating the flow of ROS during the atherogenic process. Initial studies focused on the levels of the various antioxidant enzymes known to play a role in ROS removal. One study showed that migrating smooth muscle cells and macrophages in atheromatous plaques express these enzymes intensively [40], while a different study assessed antioxidant levels in ApoE−/− mice prior to and during visible aortic lesion formation [41]. In that study, there was a coordinated increase in a wide range of antioxidant enzymes prior to visible atherogenic changes, while the expression of many antioxidant enzymes decreased during the period of lesion formation. This led these authors to suggest that the induction of antioxidant ­activities partially prevents the progression of atherogenesis, while the subsequent decline in antioxidant capacity may contribute to lesion formation. It is now increasingly recognized that knowledge of antioxidant enzyme involvement in ­limiting oxidative processes may delineate where potential therapeutic targets exist to reduce non-diabetes and diabetes-associated atherosclerosis. Indeed, overexpression of the antioxidant enzyme catalase, which removes hydrogen peroxide, reduced the severity of lesions in ApoE-deficient mice [42]. Recent attention has focused on the most abundant isoform of the glutathione peroxidase (GPx) family, glutathione peroxidase-1 (GPx1), based on a number of important clinical observations that strongly support a major role for GPx1 in limiting atherosclerosis. Blakenberg et  al. [43] first described a patient cohort where blood GPx1 activity was the strongest predictor of cardiovascular disease risk, with an inverse association between GPx1 activity and cardiovascular events. Based on this data, these authors suggested assessment of GPx1 for prognostic value in addition to that of traditional risk factors. Furthermore, they suggested that increasing GPx1 activity might lower the risk of cardiovascular events. Schnabel et  al. [44] demonstrated that plasma homocysteine (HCys) was related to future cardiovascular events in a patient cohort with coronary artery disease and that this occurred mainly in patients with low erythrocyte GPx1 activity. These authors proposed that HCys elicits its cardiovascular effect by directly affecting GPx1 activity. Winter et al. [45] found a significant association between a polymorphism in the human GPx1 gene and the risk of coronary artery disease, while Hamanishi et al. [46] found additional GPx1 polymorphisms within a diabetic population that correlated with reduced GPx1 activity and an increased risk of atherosclerosis. Recently, Nemoto et al. [47]

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reported that the presence of a Pro197Leu substitution within the GPx-1 gene may play a crucial role in determining genetic susceptibility to coronary arteriosclerosis in type 2 diabetic patients since this polymorphism was associated with increased coronary artery calcification as assessed by multi-slice computed tomography. Although in vitro and clinical data are highly supportive of a role for ROS in underpinning diabetes-associated atherosclerosis, it is only through interrogation of appropriate animal models that a true picture of the role of ROS and antioxidant defense is revealed.

2.1 Experimental Models of Diabetes-Associated Atherosclerosis with an Emphasis on Oxidative Stress 2.1.1 The GPx1 Knockout Mouse Glutathione Peroxidase-1 and Its Role in the Antioxidant Pathway Glutathione peroxidase-1 is a ubiquitously expressed antioxidant enzyme present in the cytosol and mitochondria of all living cells. One of its major functions is the second-step detoxification of hydrogen peroxide within the antioxidant pathway. A build-up of hydrogen peroxide and its subsequent non-enzymatic conversion to noxious hydroxyl radicals is prevented by the rapid interaction of GPx1 with its substrate, H2O2, and its co-factor, reduced glutathione (GSH). GPx1 is also involved in the removal of lipid peroxides [48] and it acts as a peroxynitrite reductase in the reduction of potentially damaging peroxynitrite radicals [49] (Fig.  1). In the absence of this antioxidant enzyme, a build-up of ROS ensues that are known to damage DNA, proteins and lipids [49]. GPx1 knockout (−/−) mice, generated in our laboratory [50] and by others [51, 52], have become an excellent research tool with which to establish a role for ROS in the progression and promotion of oxidant stress-mediated pathogenesis. Furthermore they have allowed us to draw meaningful conclusions about the protective role of this isoform of the GPx family of antioxidant enzymes, since standard assays do not discriminate between the different isoforms. In addition, most studies investigating the role of the GPxs do so by limiting selenium intake, which results in non-specific reductions in selenium-dependent enzymes [53], including all the selenium-dependent isoforms of GPx. The GPx1 knockout model also facilitates the distinction between the contributions of Gpx1, catalase (a peroxisomal H2O2 metabolizing enzyme) and thioredoxin peroxidase in the peroxidation of H2O2 to water. Our initial studies using this experimental model of oxidative stress showed an important role for GPx1 in the protection against ischemic-reperfusion mediated stroke [54]. In this instance, Gpx1−/− mice that were subjected to focal cerebral ischemia for 2  h via occlusion of the mid-cerebral artery showed significantly ­elevated lipid hydroperoxide levels compared with stroked control brains as well as

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NADPH Oxidase Xanthine Oxidase Cyclo-oxygenase Mitochondrial leakage dysfunctional eNOS

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Fig.  1  Removal of ROS by GPx1. The generation of superoxide radicals (−O2·) is greatly enhanced in the diabetic milieu via enzymes such as NADPH oxidase. −O2· is neutralized to water via a two-step process involving superoxide dismutase (SOD) in a first step, and glutathione peroxidase-1 (GPx1) or catalase in a second step. An imbalance in this pathway favors the build-up of hydrogen peroxide (H2O2). Fenton-type reactions occur when H2O2 or −O2· interact with transition metals such as iron (Fe2+), resulting in the production of noxious hydroxyl radicals (OH). These radicals initiate peroxidative damage to lipids, forming lipid hydroperoxides (LOOH). The functional importance of GPx1 rests in its ability to remove both hydrogen peroxide and lipid peroxides and neutralize these to water and lipid alcohol (LOH) respectively. In addition, GPx1 removes peroxynitrite radicals that form as a result of the interaction of −O2· with nitric oxide (NO). Two reduced glutathione (GSH) are consumed each time GPx1 reduces ROS, generating oxidized glutathione (GSSG)

accelerated caspase-3 activation and apoptosis of neuronal cells. Similar results were obtained in GPx1−/− mice subjected to cold-induced cerebral damage as a model of head trauma [55]. Given the strong evidence for a role for ROS in the initiation and progression of atherosclerosis, we felt that the GPx1−/− mouse model was ideally suited to studying the consequences of a lack of GPx1 on pro-atherogenic processes associated with diabetes. However, in order to understand the role of GPx1 in diabetes-associated atherosclerosis, it was important to first consider the contribution of GPx1 to proatherogenic processes not limited to the diabetic milieu. An underlying assumption would then be that the diabetic milieu, with its highly pro-oxidant environment, would facilitate even greater responses than those seen in a non-diabetic environment.

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 Px1−/− Mice Fed High Fat Diets as a Model to Study G Pro-atherogenic Mechanisms Our initial studies were performed in control mice and Gpx1−/− mice fed high fat diets (15% fat and 1% cholesterol) for 20 weeks on a C57Bl/J6 background [56]. In these animals, our biochemical analysis confirmed increased uptake of cholesterol into the vasculature of control and Gpx1−/− mice. Despite increases in nonenzymatic antioxidants after HFD-feeding (a-TOH in both plasma and vasculature, and total CoQ levels in vasculature), a rise in a-tocopheryl quinone (a-TQ), a well established marker of oxidative stress, suggested enhanced oxidative events within the vasculature of Gpx1−/− mice. However, Gpx1−/− mice failed to show an increase in aortic root lesions, nor were peroxidative events increased in plasma or aortic wall lipids compared with that seen in control animals. These results suggested that increased oxidative events within vasculature did not translate into increased lipid peroxidative damage and did not influence lipid deposition within the aortic sinus region in this strain of mice. However, the importance of GPx1 in the protection against atherosclerosis became apparent to us [57] and others [58] when the lack of GPx1 was coupled with a lack of apolipoprotein E (ApoE) in ApoE/GPx1 double-knockout (dKO) mice. ApoE−/− mice are now recommended as the murine model in which to study atherosclerosis, since these mice develop more extensive and more pathophysiologically relevant lesions throughout the aortic tree [59] that are not restricted to the aortic root as observed in high-fat-fed, non-genetically altered mice [60–62]. This allows for a more robust analysis of pathophysiologically relevant factors affecting atheroscleosis throughout the aortic tree. Furthermore, ApoE−/− mice develop atherosclerosis over a relatively short period of time as a consequence of their impaired clearance of plasma lipoproteins [63]. It should however be highlighted that LDL-R knockout mice are also a useful model in which to study pro-atherogenic processes [64], particularly since diabetes does not have as great a dyslipidemic effect in this strain of mice [19]. ApoE/GPx1 Double-Knockout Mouse Model After the establishment of our ApoE/GPx1 dKO colony (on a C57Bl/J6 background), we initially investigated the effect of a lack of GPx1 on atherosclerosis during aging. Our data showed increased lesion formation in the aortic arch and sinus region of 6- and 12-month-old female ApoE/GPx1 dKO mice fed a regular diet (4% fat) compared with age- and sex-matched ApoE−/− controls (Fig. 2a, b). In addition, Torzewski et al. [58] provided evidence that a lack of GPx1 accelerated atherosclerosis in their ApoE/GPx1 dKO mice after high fat feeding. In their study, atherosclerotic lesions were significantly increased in female ApoE/GPx1 dKO mice placed on a Western-type diet for 24 weeks. Moreover, their lesions showed increased cellularity, with an increase in macrophage content in early lesions and an increase in smooth muscle cells in advanced lesions. Furthermore, a deficiency

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a

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Fig.  2  Aortic sinus lesions, detected after staining with Oil Red O, of 6-month-old female ­ poE-deficient and ApoE/GPx1 double-knockout (dKO) mice are shown in (a). Quantitation of A lesions (b) show that sinus lesions are significantly increased in ApoE/GPx1 double-knockout mice compared with age and sex-matched ApoE-deficient controls. n = 10 mice/group. *P 96% of the GPx activity [50]. Protection against oxidative stress is therefore most likely as a result of the function of the GPx1 isoform. However, to date, no study has directly linked GPx1 to the protection against DN. Our initial studies using diabetic C57Bl/J6 GPx1−/− mice surprisingly failed to show accelerated DN [102]. Thus, the significance of a lack of GPx1 may not have been properly revealed since lipid levels are unaffected in this diabetic model. Indeed, elevated lipids may be critical in accelerating DN since clinical observations suggest that hyperlipidemia is an important contributory factor to the progression of diabetic renal disease [103, 104]. Furthermore, Lassila et al. [105] have shown accelerated nephropathy in diabetic ApoE−/− mice. Therefore, mice with both an ApoE and a Gpx1 deficiency represent a more advanced model in which to study the consequences of a lack of GPx1.

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3.1 Diabetic ApoE/GPx1−/− dKO Mouse as a Model of DN In addition to the accelerated diabetes-associated atherosclerosis detailed above, we have also detected a significant increase in characteristic markers of diabetic nephropathy in ApoE/GPx1 dKO mice rendered diabetic with STZ. This occurred against a background of elevated lipids, highlighting the involvement of both ­lipids and oxidative stress in the progression of diabetic nephropathy. Furthermore, these results emphasize the importance of using the correct experimental model to address questions of oxidative stress in diabetic nephropathy, since our previous studies in diabetic GPx1−/− mice on a C57Bl/J6 background without increased lipid involvement, failed to show significant differences [102]. In our studies using diabetic ApoE/GPx1 dKO mice, we demonstrate increased albuminuria that is associated with pathological changes that include mesangial expansion and ­upregulation of pro-fibrotic (collagen I and III, fibronectin and TGF-b) and proinflammatory mediators (VCAM-1 and MCP-1) in the diabetic ApoE/GPx1 dKO kidneys. In doing so, we establish a role for GPx1 in limiting and/or preventing diabetic nephropathy in the physiologically relevant milieu of increased lipids known to accompany diabetes [104, 105]. It is also noteworthy that lack of GPx1 caused an upregulation of collagen I and III but not type IV collagen in the diabetic kidney. Type I and III collagen are mainly expressed in the interstitial region of the kidney with some involvement in glomeruli under certain pathological situations, while type IV collagen plays a significant role in glomerular pathology [106, 107]. It is therefore likely that GPx1 plays a role in the protection of the interstitium against extracellular matrix deposition. GPx1 may therefore be of particular importance in the protection of the kidney interstitium against diabetesmediated changes, as it is now recognized that tubulointerstitial changes are a prominent feature of DN and are closely linked to the progression and ultimate decline in glomerular filtration rate [108].

3.2 Experimental Models of NADPH Oxidase-Mediated Oxidative Stress in DN It is now well established that NADPH oxidase is the main source of ROS in DN [29, 67, 69, 109]. NADPH oxidases have a distinct cellular localization in the kidney [110]. Reactive oxygen species are produced by fibroblasts, endothelial cells, ­vascular smooth muscle cells, mesangial cells, tubular cells and podocytes. NOX-1 and -4, as well as all the components that comprise the phagocytic NADPH oxidase including NOX2, are expressed in the kidney. Expression of these NOX isoforms is seen in renal vessels, glomeruli and podocytes as well as cells of the thick ascending limb of the loop of Henle, macula densa, distal tubules, collecting ducts and cortical interstitial fibroblasts [110]. Furthermore, enhanced NADPH oxidase activity has been associated with oxidative damage to DNA in diabetic glomeruli [111, 112].

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A recent study by Gorin et al. [67] has shown a role for NOX4 in DN. In this model of STZ-induced diabetes in the Sprague-Dawley rat, antisense nucleotide treatment to NOX4, administered for 2  weeks after STZ injection, reduced renal extracellular matrix accumulation and renal hypertrophy via inhibition of Akt/­ protein kinase B and ERK1/2. However, the mechanism(s) by which diabetes and high glucose activate the NADPH oxidases remain(s) speculative. It may involve a direct effect on a specific NOX isoform such as NOX4, or via other mediators such as Ang II and/or TGF-b. Thus, the renin–angiotensin system may contribute to the upregulation of NADPH oxidase-mediated ROS production in DN. Indeed, the angiotensin-converting enzyme inhibitor as well as an Ang II type 1 (AT1) receptor blocker were able to inhibit the increase in ROS generation and reduce NAD(P)H oxidase subunit p47phox protein expression in rats with type 1 diabetes [113]. As mentioned previously, NOX1 and 2 knockout mice are now available to delineate the precise contribution of the various NOX isoforms to the development of DN and are expected to highlight the significance of NADPH oxidase as a ­possible new therapeutic target in the reduction and/or prevention of DN [70].

4 Diabetic Cardiomyopathy Extensive clinical and experimental evidence has substantiated the existence of a specific diabetes-associated cardiac disease, characterized by structural, functional and metabolic changes of the heart, in the absence of vascular complications [4]. These cardiomyopathic changes cannot be explained solely by poor coronary perfusion. Structural changes include left ventricular hypertrophy (LVH) and/or cardiac interstitial fibrosis [114, 115]. In particular, increased muscle mass indicative of myocardial hypertrophy, as well as cavity dilation and depressed ventricular performance consistent with impaired cardiac function, have been reported in diabetic subjects [115]. Large clinical studies of diabetic patients support this notion, e.g., the Framingham Heart Study, which demonstrated an association between diabetes and increased left ventricular (LV) wall thickness and increased heart mass [116]. These clinical findings have been confirmed in experimental models where an increase in wall thickness and LV dimensions were observed in diabetic rats and these changes were accompanied by cardiac dysfunction [114, 117]. Moreover, LV-weight to body-weight ratio, a marker of LVH, was higher in diabetic rats compared with non-diabetic control rats [114]. Histopathological changes include diffuse myocardial fibrosis [118] with changes in the myocardial extracellular matrix [119]. In particular, accumulation of myocardial collagen types I, III and VI have been reported. The increase in type I and III collagen may in turn contribute to altered cardiac hemodynamics due to increased stiffness and loss of elasticity. Furthermore, the increase in type III and VI collagen may impede the exchange of cellular substrates, which has been suggested to increase oxidative stress [119]. Oxidative stress has been implicated in the progression toward overt diabetic heart failure and has recently been reviewed by Mellor et  al. [120]. Evidence

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implicating ROS in diabetic cardiomyopathy include increased cardiac lipid ­peroxidation [121, 122] and elevated oxidized glutathione in diabetic heart tissue [7]. Recently superoxide production was shown to increase in the diabetic Zucker obese rat via a mechanism that includes glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH [123]. Strong evidence in support of a role for ROS in the development of cardiomyopathy is also evident under non-diabetic conditions. Indeed, the chronic release of ROS has been linked to the development of LVH and progression to heart failure [13], impaired calcium handling and the stimulation of myocytes to produce more ROS [124]. Furthermore, abnormal activation of NADPH oxidase in response to angiotensin II, noradrenaline and tumor necrosis factor-a contributes to cardiac myocyte hypertrophy [13]. In addition, fibrosis and collagen deposition involved in the remodeling of the failing myocardium are dependent on ROS release [13]. Intervention studies using various antioxidants have provided further evidence in support of a role for ROS in cardiomyopathy. For example, vitamin E therapy delayed the development of heart failure, improved the redox state and reduced lipid peroxidation in a guinea pig model of ascending aortic constriction [125]. In addition, in animals with vitamin E deficiency, myopathy of the heart muscle is frequently observed [125]. Strong evidence for the protective role of antioxidants in diabetic cardiomyopathy comes from diabetic OVE26 mice specifically overexpressing the potent antioxidant protein metallothionein in their cardiomyocytes [124, 126]. These mice, which normally exhibit extensive cardiomyopathy, showed reduced cardiac damage and improved cardiomyocyte survival. Furthermore, mice overexpressing the antioxidant enzyme GPx1, albeit in a non-diabetic model, ­display improved LV function through the inhibition of LV remodeling, as well as reduced cardiomyocyte apoptosis and interstitial fibrosis compared with wild-type mice [127]. Two further studies have investigated the functional significance of GPx1 in the protection of the myocardium during ischemia/reperfusion injury. Mice lacking GPx1 were shown to be more susceptible to ischemia/reperfusion injury [52] while conversely, mice overexpressing GPx1, were protected from such injury [128]. Similarly, overexpressing the antioxidant enzyme catalase preserved normal cardiac morphology and prevented contractile defects in diabetic OVE26 mice [129], suggesting an important role for peroxide-reducing antioxidants in the protection against diabetic cardiomyopathy. In addition, MnSOD has been shown to play an important role in maintaining normal heart function. MnSOD-deficient mice die from cardiomyopathy, neurodegeneration and metabolic acidosis within the first 10 days of life [130]. Interestingly, Cu/ZnSOD knockout mice exhibit a less severe phenotype than that of MnSOD-deficient mice, where Cu/ZnSOD knockout mice are essentially normal but more susceptible to neuronal injury [131]. It is presently thought that myocyte apoptosis is a major contributory component to the progression of diabetic cardiomyopathy [13, 132, 133]. Cell death is an important cause of various myocardial abnormalities – in particular, loss of contractile tissue, compensatory hypertrophy of myocardial cells and fibrosis [133]. However the mechanisms leading to diabetic myocardial apoptosis remain unclear [133]. ROS are powerful mediators of apoptosis [13, 132]. Indeed, Cai et al. [133]

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showed that treatment with high glucose caused an increase in ROS production as  well as increased apoptosis of cardiac myoblasts and suggested that the ­hyperglycemia-induced apoptosis occurred via ROS-mediated mechanisms [133]. Furthermore, treatment of these cardiomyocytes with superoxide or H2O2 induced apoptosis via different apoptotic pathways, implying that there are several redoxsensitive apoptotic mechanisms in cardiac myocytes. Mitochondrially derived ROS have been proposed to contribute to diabetic cardiomyopathy. Evidence for this was shown in a recent study by Song et  al. [134] who used the OVE26 mouse model of severe type 1 diabetes to measure myocyte contractility and the source of ROS generation via a mitochondrial specific ROS detection system. Initial contractility was impaired in their diabetic myocytes and ROS production was elevated after exposure to either high glucose or angiotensin II (AngII). In particular, superoxide, detected via the mitochondrial sensor MitoSOX Red, confirmed that mitochondria are a major source of ROS in these diabetic myocytes. These authors speculate that the AngII-induced increase in mitochondrial ROS occurs via a pathway that includes cytoplasmic superoxide derived from NADPH oxidase. This assumption is based on the fact that AngII acts on the AT1 cell surface receptor and not on mitochondria, implying that a pathway exists linking the cardiomyocyte AngII receptor to mitochondrial generation of ROS. Such a pathway has also been proposed by Zhang et al. [135] who suggest that cytoplasmic superoxide, derived from NADPH oxidase, induces the opening of mitochondrial KATP channels, thereby increasing mitochondrial ROS production. In the diabetic heart, NADPH oxidase is a likely mediator of this effect since it is present in the heart [136], it is upregulated by diabetes [137, 138] and, furthermore, hyperglycemia [138] and AngII [135] are known inducers of NADPH oxidase-mediated ROS. This mechanism of cytosolic ROS generation leading to increased mitochondrial ROS may also occur in other diabetic complications such as diabetic nephropathy. Evidence comes from recent data of Coughlan et al. [139] who show that AGEs lead to increases in cytosolic ROS, which facilitate the production of mitochondrial superoxide. Inhibiting cytosolic ROS production with apocynin or lowering AGE concentration with the AGE-crosslink breaker alagebrium prevented the production of mitochondrial ROS. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in RAGE knockout mice [139]. Our laboratory has investigated whether lack of the antioxidant enzyme GPx1 affects known markers of diabetic cardiomyopathy in C57Bl/J6 GPx1−/− mice made diabetic with STZ (Fig.  3a). In our model, elevated levels of H2O2, fatty acid hydroperoxides and peroxynitrite occur as a consequence of the lack of their removal by the second step antioxidant enzyme GPx1. Thus, Gpx1 knockout mice are a useful model to address the role of GPx1 and the involvement of these particular ROS in diabetic cardiomyopathy. In this model, we detected significantly increased picrosirius red staining after 3 months of diabetes in diabetic GPx1−/− hearts compared with diabetic controls (Fig. 3b), suggesting that fibrosis is enhanced by the lack of GPx1. After 6 months of diabetes, we observed a significant increase in the gene expression of the cardiac hypertrophic peptide, atrial natriuretic peptide (ANP) (Fig. 3c),

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Fig. 3  Diabetic GPx1 knockout mice hearts display features of accelerated diabetic cardiomyopathy. Despite achieving equivalent blood glucose levels of approximately 20 mM as shown in 3a, diabetic GPx1 knockout hearts show significantly increased percent fibrosis as shown in 3b (*P