Oxidative Stress and Antioxidants: Their Role in Human Disease

OXIDATIVE STRESS AND ANTIOXIDANTS: THEIR ROLE IN HUMAN DISEASE No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

OXIDATIVE STRESS AND ANTIOXIDANTS: THEIR ROLE IN HUMAN DISEASE

RAMON RODRIGO EDITOR

Nova Biomedical Books New York

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Rodrigo, Ramon. Oxidative stress and antioxidants : their role in human disease / Ramon Rodrigo. p. cm. Includes bibliographical references and index. ISBN 9781607415541 1. Oxidative stress. 2. Antioxidants. I. Title. RB170.R636 2009 616.07--dc22 2009013526

Published by Nova Science Publishers, Inc.  New York

Contents Preface

vii

Chapter I

Oxidative Stress: Basic Overview Joaquin Toro and Ramón Rodrigo

Chapter II

Hypertension Ramón Rodrigo

25

Chapter III

Atherosclerosis Víctor Molina and Ramón Rodrigo

63

Chapter IV

Postoperative Atrial Fibrillation José Vinay and Ramón Rodrigo

91

Chapter V

Acute Renal Failure Joaquín Toro, Víctor Molina and Ramón Rodrigo

111

Chapter VI

Pre-Eclampsia Mauro Parra

135

Chapter VII

Metabolic Syndrome Rodrigo Castillo

159

Chapter VIII

Diabetes Mellitus Rodrigo Castillo

193

Chapter IX

Nonalcoholic Steatohepatitis Juan Gormaz and Ramón Rodrigo

223

Chapter X

Neurodegenerative Disorders Rodrigo Pizarro

257

Chapter XI

Glaucoma Leonidas Traipe, Rodrigo Castillo and Ramón Rodrigo

297

Index

1

321

Preface Oxidative stress is a relatively new concept that has been widely implicated in biomedical sciences during the last 20 years. It significantly participates in the pathophysiology of highly prevalent diseases such as diabetes, hypertension, preeclampsia, atherosclerosis, acute renal failure, Alzheimer and Parkinson diseases, among others. The metabolism of oxygen by cells generates potentially deleterious reactive oxygen species (ROS). Under normal conditions the rate and magnitude of oxidant formation is balanced by the rate of oxidant elimination However, an imbalance between pro-oxidants and antioxidants results in oxidative stress. Increased ROS levels in the cell have a substantial impact either leading to defective cellular function, aging, or disease. Therefore, a better understanding of the roles of ROS-mediated signaling in normal cellular function as well as in disease is necessary for developing therapeutic tools for oxidative stress-related pathologies. The potential beneficial role of antioxidants is discussed in the light of experimental studies, as well as clinical trials aimed to determine the outcome of patients. “Oxidative Stress and Antioxidants: Their Role in Human Disease” is a practical guide for pathophysiology of oxidative stress and the latest therapeutic advances to modulate the antioxidant defense. This includes evidence from clinical trials, regarding the use of antioxidants and preconditioning, to protect the organism against ROS. Chapter I - Over the last decades, a new concept involving the biological effects of highly reactive oxygen and nitrogen species in the mechanisms causing disease has filled the scientific journals. These reactive species, mainly free radicals, are found in normal physiological condition and can be beneficial when produced at low levels. However, they are harmful at high concentrations when the endogenous antioxidant defense systems are overwhelmed (oxidative stress), what has been implicated in disease states. This paradigm has been widely documented for many settings, and a causal relationship has been suggested for oxidative stress and some highly prevalent human pathologic alterations, such as atherosclerosis, hypertension, preeclampsia, diabetes, among others. This chapter provides a comprehensive review of the basis for the biochemical and physiological mechanisms involving reactive oxygen species generation and depuration, as well as their effects on biological molecules. In addition, the defensive response of the antioxidant defense system in vivo against oxidative damage is also analyzed. Nevertheless, before extensively examining the specific mechanisms hypothesized for the diverse

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pathologies, it is included a basic review of the biochemistry accounting for the alterations mediated by oxidative stress. Chapter II - Reactive oxygen species (ROS) and reactive nitrogen species play a key role in the modulation of the vasomotor system. Thus, ROS are recognized as mediators of the vasoconstriction induced by angiotensin II, endothelin-1 or urotensin-II, among others; while nitric oxide (NO) is a major vasodilator. In physiological conditions, low concentrations of intracellular ROS play an important role in normal redox signaling involved in maintaining vascular function and integrity. In addition, under pathophysiological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. The fact that ROS play a key role in development of hypertension is supported by the findings of increased production of superoxide anion and hydrogen peroxide, reduction of NO synthesis, and a decrease in bioavailability of antioxidants in human hypertension. In both animal models and humans, increased blood pressure has been associated with an excessive endothelial production of ROS (oxidative stress) which may be both a cause and an effect of hypertension. Antioxidants, whether synthesized endogenously or exogenously administered, are reducing agents that neutralize these oxidative compounds before they can cause damage to biomolecules. In the management of hypertension and other cardiovascular diseases, the primary interest was focused on the therapeutic possibilities of antioxidants to target ROS, thus avoiding hypertensive end-organ damage. The use of antioxidant vitamins, such as vitamin E and vitamin C, has gained considerable interest for their role as protecting agents against vascular endothelial damage, in this way contributing to ameliorate chronic diseases, beyond its essential function associated to body deficiencies. However, promising findings from experimental investigations, the results of clinical trials aimed to demonstrate antihypertensive effects of antioxidant supplementation are disappointing. Nevertheless, the methodology used in some of these studies makes them a matter still to be debated. Some studies reported a potential antihypertensive effect, particularly when using association of two or more antioxidants. Even more, antioxidant diets low in fat, have found to be of most significant benefit in hypertensive patients. Taken together data are consistent with the view that while an antioxidant alone has not yet demonstrated its efficacy as a therapeutic antihypertensive agent, the synergistic actions among the various antioxidants appear to be effective to counteract the ROS effect on the vascular wall. These effects could arise from their complex biological actions, from their ability not solely to scavenge ROS, but also to prevent their formation through down regulation of NADPH oxidase and up-regulation of endothelial NO synthase and antioxidant enzymes. Chapter III - Atherosclerosis is a major source of mortality, being the underlying cause for most cases of cardiovascular diseases such as ischemic heart disease and cerebrovascular disease. Reactive oxygen species (ROS) can regulate several cellular processes, having a key role in the homeostasis of the vascular wall. There is compelling evidence pointing to ROS as important factors for the development of atherosclerosis. Many of the proatherogenic actions of ROS occur through the generation of oxidized LDL. Also, ROS can contribute to the development of endothelial dysfunction through the consumption of nitric oxide and generation of peroxynitrite. Endothelial dysfunction constitutes an early feature of

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atherogenesis, preceding the alterations that later perpetuate the lesion formation. Atherogenesis includes several processes, such as accumulation and oxidation of LDL in the subendothelial space, expression of adhesion molecules and chemoattractant mediators, adhesion of monocytes, generation of foam cells, production of inflammatory mediators and proliferation of certain cell types. Since most of these processes can be modulated by ROS, supplementation with antioxidants is expected to exert some degree of protection against atherosclerosis. Several lines of evidence support a role of antioxidant supplementation in attenuating some of the processes involved in atherogenesis. However, clinical trials have failed to consistently prove a protective effect. The potential role of antioxidant supplementation against atherosclerosis development or progression remains an open question. Chapter IV - Atrial fibrillation is an arrhythmia occurring frequently within the first few days in 10% to 65% of patients after major cardiothoracic surgery (postoperative atrial fibrillation, POAF). It is associated with increased morbidity and mortality and longer, more expensive hospital stays. Despite the use of strategies to prevent POAF through the prophylactic use of agents such as β-adrenergic blockers, amiodarone, or others, a considerable percentage of the patients still presents the arrhythmia. The involvement of oxidative stress in the mechanism of POAF is supported by an increasing body of evidence indicating that the formation of reactive oxygen species (ROS) released following extracorporeal circulation are involved in the structural and functional myocardial impairment derived from the unavoidable ischemia–reperfusion cycle of this setting. ROS behave as intracellular messengers mediating pathological processes, such as inflammation, apoptosis and necrosis, thereby participating in the pathophysiology of POAF. Consequently, myocardial electrical and structural remodeling associates with the appearance of functional impairment consistent with alterations in electrical conduction. Therefore, it seems reasonable to assume that the reinforcement of the antioxidant defense system should protect the heart against functional alterations in the cardiac rhythm in this setting. Interestingly, exposure to low to moderate doses of ROS could trigger a cellular defensive response characterized by a prevailing effect of survival over apoptotic pathway, what should be considered a therapeutic target. The present chapter examines the molecular basis accounting for the contribution of oxidative stress to the development of POAF. In addition, it is presented the clinical and experimental evidence to support a new paradigm based in the prophylactic reinforcement of the antioxidant defense system toward reduction in the susceptibility of cardiomyocytes to ROS-induced injury. Chapter V - Acute renal failure (ARF) is a condition characterized by a rapid decrease in renal function, leading to an imbalance in water and solutes metabolism. It constitutes a major cause of morbidity and mortality in hospitalized patients worldwide, mainly in elderly population. Despite the medical advances, over the past fifty years the mortality of ARF has not diminished. This is often attributed to increased risk factors prevalence, mainly those derived from changes in our lifestyle. However, it is also possible that the therapeutic methods used until these days are not aiming on the right direction, probably due to lack of knowledge about some of the mechanisms leading to the development and progression of ARF.

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Over the last decades a large body of evidence has emerged supporting a role of oxidative stress in the pathogenesis of a variety of diseases, including ARF. Indeed, both reactive oxygen and nitrogen species are thought to enhance tubular damage caused from either renal ischemia or direct toxic injury. Nevertheless, the role of oxidative stress in ARF pathogenesis has not been fully established and some evidence is even contradictory. A better understanding regarding the real contribution of oxidative stress to ARF development and progression is required for the design of potentially preventive interventions, such as antioxidant supplementation. Indeed, clinical trials on this matter have been carried out with promising results. This chapter presents an update of the current evidence supporting a role of oxidative stress in ARF pathophysiology, and the potential role of antioxidants in the prevention and treatment of this disease. Chapter VI - Pre-eclampsia (PE) is the most important complication of human pregnancy worldwide and a major contributor to maternal and fetal morbidity and mortality. It is a disease of two stages. The first stage concerns the relative failure of early trophoblast invasion and remodeling of the spiral arteries, leading to a poor blood supply to the fetoplacental unit, exposing it to oxidative stress. The second stage is characterized by maternal endothelial dysfunction, leading to the clinically recognized symptoms of the syndrome, which include hypertension, proteinuria, thrombocytopenia and impaired liver function. Furthermore, the modification of spiral arteries occurs during the first and early second trimester of pregnancy, leading to uteroplacental hypoperfusion and fetal hypoxia. Despite much work in the last decade, the causes that trigger PE are uncertain and the predictive value of potential risk factors is poor. Increasing evidence suggests that placental and systemic oxidative stress plays a crucial role in its development. Indeed, oxidative stress and disrupting angiogenesis is considered the link bridging the two stages of the disease. Markers of oxidative stress in women with established PE have shown both increased lipid peroxidation in placental tissue, along with increased in maternal plasma biomarkers indicating decreased antioxidant capacity and increased lipid peroxidation. These findings have contributed to the interest in using antioxidants to prevent the development of PE. The lack of appropriate early predictors of the disease has determined that the risk groups for primary prevention of PE should be characterized on the basis of the clinical history of the patients and from knowing that is possible to establish some risk factors. A large number of publications suggest a potential role of antioxidant nutrients in the prevention of PE in women at high increased risk of the disease. Vitamins C and E have been the main antioxidants agents used for this purpose. Despite the biological properties of these compounds, exerting ROS scavenging and a down-regulation of ROS, the results of clinical trials do not support benefits for routine supplementation with vitamins C and E during pregnancy to reduce the risk of PE. This chapter examines the role of oxidative stress in the pathophysiology of PE and reviews the available data on the use of antioxidant compounds, mainly vitamins C and E, to prevent the development of this disease. Chapter VII - The biochemical steps linking insulin resistance with the metabolic syndrome have not been completely clarified. Mounted experimental and clinical evidence indicates that oxidative stress is an attractive candidate for a central pathogenic role since it

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potentially explains the appearance of all risk factors and supports the clinical manifestations. Indeed, metabolic syndrome patients exhibit activation of biochemical pathways leading to increased delivery of ROS, decreased antioxidant protection and increased lipid peroxidation. The described associations between increased abdominal fat storage, liver steatosis and systemic oxidative stress, the diminished concentration of nitric oxide derivatives and antioxidant vitamins, and the endothelial oxidative damages observed in subjects with the metabolic syndrome support oxidative stress as the common second-level event in an unifying pathogenic view. Moreover, it has been observed that oxidative stress regulates the expression of genes governing lipid and glucose metabolism through activation or inhibition of intracellular sensors. Diet constituents can modulate redox reactions and the oxidative stress extent, thus also acting on nuclear gene expression. As a consequence of the food–gene interaction, metabolic syndrome patients may express different disease features and extents according to the different pathways activated by oxidative stress-modulated effectors. This view could also explain family differences and interethnic variations in determining risk factor appearance. Chapter VIII - Elevation of glycemia in diabetic patients may lead to the autooxidation of glucose, glycation of proteins, and the activation of polyol metabolism. These changes accelerate the generation of reactive oxygen species (ROS) and increase oxidative modification of lipids, DNA, and proteins in various tissues. Thus, oxidative stress occurring in this setting may play an important role in the development of the chronic complications of diabetes, such as nephropathy, neuropathy, and lens cataracts. Langerhans islets are more vulnerable to the occurrence of oxidative stress, since they contain low levels of antioxidant enzyme activities compared to other tissues. High glucose concentrations are known to give rise to a manifestation named glucose toxicity. Major manifestations of glucose toxicity in the pancreatic β-cells are defective insulin gene expression, diminished insulin content, and defective insulin secretion. The link between the clinical complications and oxidative stressrelated parameters has been established by the study of advanced glycation end products (AGEs). Among the latter, heterocyclic amines, acrylamide, and AGEs are well-known compounds hypothesized to cause harmful health effects. First, AGEs act directly to induce cross-linking of long-lived proteins, such as collagen, to promote vascular stiffness, thus altering the structure and function of vasculature. Second, AGEs can interact with their receptors to induce intracellular signaling leading to enhanced oxidative stress and elaboration of key proinflammatory and prosclerotic cytokines. Over the last decade, a large number of preclinical studies have been performed, targeting the formation and degradation of AGEs, as well as their interaction with specific receptors. Translational research with humans is now under way to ascertain whether this protection can be provided to patients experiencing inadequate glycemic control. Chapter IX - Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver diseases characterized mainly by macrovesicular steatosis that occurs in the absence of alcoholic consumption. NAFLD is closely associated with comorbid conditions, such as obesity, dyslipidemia, and insulin resistance. It is a medical condition in which the liver is invaded with fat and excessive amounts of lipids are present within hepatocytes. There is increasing evidence to consider that fatty liver is the hepatic manifestation of the metabolic syndrome, a growing problem in the modern western world. NAFLD might worsen into a

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more serious condition, known as nonalcoholic steatohepatitis (NASH), in which fat accumulation is accompanied by an inflammatory process in the liver. The clinical relevance of these conditions is given by the high prevalence of NAFLD in the general population and to the possible evolution of NASH towards end-stage liver disease, including hepatocellular carcinoma, as well as the need for liver transplantation. The molecular mechanism whereby NASH might eventually lead to fibrosis, and severe cirrhosis in some patients, is a process associated with increased production and release of inflammatory mediators, such as nitric oxide (NO), cytokines, and reactive oxygen species (ROS) by the cells. Oxidative stress caused by increased ROS plays an important role in the pathogenesis of NASH. These reactive species would derive from mitochondria, cytochrome P-450 2E1, peroxisome, and iron overload in the liver with steatosis. Excessive ROS is considered to cause simple steatosis to progress to NASH. Regardless the origin of hepatic fat, it could produce a rise of hepatic free fatty acids. The latter, particularly the polyunsaturated ones, are closely linked to ROS generation by different pathways, including increased oxidation in different cellular organelles, disruption of mitochondria and endoplasmic reticulum, microsomal cytochrome P450 activation and ceramide formation. In addition, increased ROS production could derivate not only in hepatocyte cell death but also in the activation of liver resident cells, such as Kupffer, stellate and endothelial cells. This might enhance the original oxidative stress, inflammatory response and subsequent immune infiltration thus aggravating NASH. Up to date no absolute effective medical treatment is available for NASH patients. Therapy is predominantly aimed at controlling the comorbid conditions, such as obesity, insulin resistance, and dyslipidemia. However the major role of oxidative stress in the pathogenesis of NASH suggests that the antioxidant treatment would be an effective therapy. Hence, both several substances with different antioxidants mechanism and effects related with the redox balance have been assayed in small clinical trials. These agents have shown the ability of improve the outcome of patients, thus opening the door to new strategies to manage or treat this disease. This chapter provides the clinical and experimental evidence to support the role of oxidative stress in the pathophysiology of NAFLD and NASH, as well as the molecular bases promoting the development of mechanism-based therapeutic interventions, mainly clinical trials aimed to target specific pathways involved in the pathogenesis of NASH. Chapter X - Oxidative stress has been related to the pathogenesis of virtually every neurodegenerative disease. However, two clinical entities stand out for the major epidemiological burden they provide, as they are intimately related to our increasingly aging population. Alzheimer’s disease and Parkinson’s disease are the two most prevalent neurodegenerative diseases affecting roughly over 5.5 million people in the United States alone. In spite of this, the understanding of their underlying pathophysiological mechanisms is scarce, and thus, they have remained difficult to treat, prevent or cure. Early diagnosis is fundamental, as is in early stages of the neurodegenerative process when therapeutic interventions in both animal models and clinical trials have proven more beneficial. However, early detection can be a painstaking procedure due to their subtle, highly unspecific first clinical features and still rather undeveloped biomarkers. It is in all of these tasks where the understanding of the roles of oxidative stress in the pathogenesis of such

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conditions has been proved beneficial. Biochemical markers of oxidative stress now seem a feasible way of early detection of such diseases. We could also give possible explanations for the mixed results obtained in clinical trials using antioxidant supplementation against neurodegenerative diseases. The task now is to continue looking deeper at oxidative stress in the mechanisms of such diseases, but also to develop new therapeutic resources to meet the needs of a rapidly growing population. This chapter deals with the general pathophysiology of oxidative stress and its role in the pathogenesis and antioxidant supplementation in the detection, understanding and treatment of Alzheimer’s disease and Parkinson’s disease. Chapter XI - Glaucoma constitutes an increasingly serious public health problem, moreover in developed countries and is an important cause of blindness after cataracts. It is an optic neuropathy that implies loss of retinal ganglion cells, including their axons, and a major tissue remodeling, especially in the optic nerve head. Although increased intraocular pressure is a major risk factor for glaucomatous optic neuropathy, there is little doubt that other factors such as ocular blood flow play a role as well. Mechanisms leading to glaucomatous optic neuropathy are not yet clearly understood. There is, however, increasing evidence that both activation of glial cells and oxidative stress in the axons play an important role. The involvement of reactive oxygen species (ROS) in the pathogenesis of glaucoma is supported by various experimental findings, including: (i) resistance to aqueous humor outflow is increased by hydrogen peroxide by inducing trabecular meshwork (TM) degeneration; (ii) TM possesses remarkable antioxidant potential, mainly explained by superoxide dismutase and catalase activities and glutathione pathways, all that is found decreased in glaucoma patients; and (iii) intraocular-pressure increase and severity of visualfield defects in glaucoma patients paralleled by the amount of oxidative damage of DNA affecting TM. Vascular alterations, which are often associated with glaucoma, could contribute to the generation of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal cells, appears to be involved in the neuronal cell death affecting the optic nerve in glaucoma. Despite the major pathogenic role of ROS in the pathophysiology of glaucoma, clinical trials testing the efficacy of antioxidant drugs for its management are still lacking.

In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter I

Oxidative Stress: Basic Overview

1

Joaquin Toro1 and Ramón Rodrigo2

Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile. 2 Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile Supported by FONDECYT, grant 1070948

Abstract Over the last decades, a new concept involving the biological effects of highly reactive oxygen and nitrogen species in the mechanisms causing disease has filled the scientific journals. These reactive species, mainly free radicals, are found in normal physiological condition and can be beneficial when produced at low levels. However, they are harmful at high concentrations when the endogenous antioxidant defense systems are overwhelmed (oxidative stress), what has been implicated in disease states. This paradigm has been widely documented for many settings, and a causal relationship has been suggested for oxidative stress and some highly prevalent human pathologic alterations, such as atherosclerosis, hypertension, preeclampsia, diabetes, among others. This chapter provides a comprehensive review of the basis for the biochemical and physiological mechanisms involving reactive oxygen species generation and depuration, as well as their effects on biological molecules. In addition, the defensive response of the antioxidant defense system in vivo against oxidative damage is also analyzed. Nevertheless, before extensively examining the specific mechanisms hypothesized for the diverse pathologies, it is included a basic review of the biochemistry accounting for the alterations mediated by oxidative stress.

1. Introduction The oxidation and reduction reactions in biological systems (redox reactions) represent the basis for numerous biochemical mechanisms of metabolic changes [1]. In biological

Joaquin Toro and Ramón Rodrigo

2

systems, instead of using the terms reducing and oxidant agent, it is more frequent to use the denominations of antioxidant and pro-oxidant, respectively [2]. A reducing agent, or antioxidant, is a substance which donates electrons, whereas an oxidant, or pro-oxidant agent, is a substance that accepts electrons. Cells are constantly exposed to oxidants from both physiological processes, such as mitochondrial respiration [3] and pathophysiological conditions such as inflammation, foreign compound metabolism, and radiation among others [4]. Oxidative stress constitutes a unifying mechanism of injury of many types of disease processes. This alteration is encountered when there is an imbalance between the production of reactive oxygen species (ROS) and the ability of the biological system to readily detoxify these reactive intermediates or easily repair the resulting damage. Reactive oxygen species are a family of highly reactive species that can be beneficial, as they are used by the immune system as a way to attack and kill pathogens. Nevertheless, when these species are found in excess they might cause cell damage either directly or working as intermediates in diverse signaling pathways. Reactive nitrogen species (RNS) may have also a similar behavior: While nitric oxide radical (NO) has vasorelaxing and antiproliferative properties, peroxynitrite anion (ONOO-) increases intracellular ROS concentration, with deleterious consequences. This chapter deals first with the basis of physiological mechanisms of ROS generation, and subsequently with the explanation of their role as toxic molecules in diverse pathophysiological conditions leading to several common diseases. In addition, the components of the antioxidant defense system in vivo are described.

2. Oxidative Stress 2.1. Background The first radical oxygen was discovered by Linus Pauling in 1930s and it was described as superoxide [5]. Pauling had no knowledge that this radical could be produced biologically or that it could also be the core of several many disease processes. In the same decade, Mann and Keilin [6] purified the superoxide dismutase (SOD) protein from bovine blood and liver, as a copper-binding protein of unknown function. The protein was called “erythrocuprein” or “hepatocuprein” or later “cytocuprein.” The purification was based solely on copper content. Until late 1960s, the pathophysiological importance of ROS was completely unknown. However, several new findings would dramatically lead to a change of this situation: 1. The discovery of McCord and Fridovich in 1968-1969 described the enzymatic activity of the erythrocyte SOD, which led to eliminate the “Pauling free radical”, or superoxide anion (O2•–) terminology, and in the same year it was found that SOD was contained in almost all mammalian cells [7, 8]. The latter finding suggested that O2•– was a physiological product. 2. In 1969, Knowles et al. showed that the enzyme xanthine oxidase (XO) could indeed produce superoxide [9].

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3. In 1973 Babior et al. [10] showed that the bactericide action of the neutrophil was associated with large amounts O2•– generation, thereby linking the inflammation process to ROS generation. It was apparent that some of the tissue damage associated with the inflammatory process could be attributed to neutrophil-generated O2•–, and herein SOD would protect cells and extracellular components from damage [11, 12]. 4. In 1980, the discovery of the endothelium-derived relaxing factor allowed formulation of a novel concept in the pathogenesis of hypertension [13]. Nevertheless, it took long seven years to determine the identity of this factor and to accept that it corresponds to NO [14, 15]. 5. In 1981, Granger et al. [16] showed that tissue damage of ischemia/reperfusion in cat intestine was caused by increased ROS generation. From those years until now, hundreds of further researches were needed to achieve our present knowledge on how oxidative stress is implicated in diverse and seemingly unrelated diseases.

2.2. Generation of Reactive Oxygen and Nitrogen Species The generation of ROS is a physiological and normal attribute of any kind of aerobic life. In mammalian, under physiological conditions, cells metabolize approximately 95% of the oxygen (O2) to water, without formation of any toxic intermediates. Water if formed according to the following tetravalent reaction: O2 + 4H+ + 4e– → 2H2O The first impressions about oxygen as an element were made by the Swedish researcher C.W. Scheele in the XVIII century. However, it was only in XX century when it was demonstrated what Scheele himself had already anticipated that O2 in its pure state at high pressure and concentration is toxic for animals, and herein for several life forms. The later was followed by new interesting discoveries, generating the controversy called until these days as “the oxygen paradox” Several investigations from the last thirty years were needed to agree that, in normal conditions, a minimal 5% of O2 is metabolized through univalent reduction, following four different reactions or stages: Reaction 1: Reaction 2: Reaction 3: Reaction 4:

O2 O2•– H2O2 •OH

+ + + +

e → O2•– e → H2O2 e → •OH e → H2O

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Indeed, the final product is still H2O. However, through these four reactions three highly toxic species are formed, two of them being free radicals: O2•– and hydroxyl radical (•OH). Hydrogen peroxide (H2O2) is still a highly reactive compound, but not a radical in strict sense. This four stages model was the first to be discovered, and in fact it explains in general terms the mitochondrial generation of ROS in normal cellular metabolism. The intermediates do not leave the complex before the process is finished, but in some pathophysiological conditions ROS can leave the respiratory burst. On the other hand, once synthesized, NO might follow different pathways:

1. Diffusion to neighbor cells. The presence of an unpaired electron on its molecule allows NO to interact with transition metals, derived from different enzymes, to modulate its activity [17, 18]. The diffusion coefficient of this gas depends on lipids and proteins on its microenvironment. 2. Autooxidation: Usually it occurs at severe high concentrations of NO. In the presence of O2 it becomes into dinitrogen trioxide (N2O3) [19, 20]. This reaction increases when it takes place in hydrophobic sites, such as the inside part of lipid membranes or proteic nucleus [20, 21]. The molecule of N2O3 is a powerful nitrosant agent, with great affinity for nucleophilic sites [18]. 3. Reaction with superoxide. The half-life of NO and therefore its biological activity is decisively determined by O2•– concentration [22]. This reaction has a limiteddiffusion kinetic curve, and thus it is thought that it rules the destination of NO in the presence of O2•– [23, 24]. The final product is ONOO-, a highly oxidant RNS similar to •OH in terms of toxicity. Therefore, ONOO- formation represents a major potential pathway of NO reactivity, depending on the rates of tissue O2•– production. In mammalian cells ROS might be formed through different pathways, either enzymatically or non-enzymatically. For instance, the generation of O2•–, as well as other ROS, requires cell activation involving alteration of the cell membrane structure what in turn activates the generation of lipid peroxidation product molecules. In the context of this chapter, relevant pathways will be described below. a.

Fenton reaction. This reaction has been known since 1894 and is currently one of the most powerful oxidizing reactions available. The reaction involves H2O2 and a ferrous iron catalyst. The peroxide is broken down into a hydroxide ion and a •OH. The latter is the primary oxidizing species and can be used to oxidize and break apart organic molecules.

Fe(II) + H2O2 → Fe(III) + •OH + −OH) [25]. It is well known that organic compounds can be easily oxidized. One primary advantage of the Fenton's Reaction is that it does not produce further organic compounds or inorganic solids such as permanganate and dichromate, since there is no carbon in the peroxide. This

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makes the Fenton's Reaction more appealing than a biological process, if the goal is removal of organic compounds. The mechanism of reaction with respect to hydrogen peroxide is very complex and may change with conditions of the reaction. b. Haber-Weiss reaction: The one-electron reduction of hydrogen peroxide by superoxide has also been invoked as a potential source of •OH: O2•− + H2O2 → O2 + •OH + OH− This scheme has been exhaustively investigated and it is now generally accepted that the Haber-Weiss reaction does not occur in the absence of metal catalysis. This reaction combines a Fenton reaction and the reduction of Fe(III) by O2•–, yielding Fe(II) and O2 Fe(III) + O2•− → Fe(II) + O2 [26].

c. Xanthine oxidase: The enzyme xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid, generating O2•−. This enzyme plays an important role in the catabolism of purines in some species, including humans. Under pathological conditions, such as tissue ischemia, xanthine dehydrogenase can be converted to XO. xanthine + H2O + O2 → uric acid + O2•−

d. NADPH oxidase: The enzyme NADPH oxidase (Nox) catalyzes the one electron reduction of O2 to generate O2•−, using NADPH as the source of electrons. This enzyme has a complex function that is most easily understood in the context of the activated neutrophil, wherein it generates large amounts of toxic superoxide anion and other ROS important in bactericidal function. In addition, it is also functional in membranes of vascular endothelial and VSMC, and fibroblasts providing a constitutive source of O2•−. This enzyme consists of several membrane-bound subunits (gp91, Nox, and p22phox) and cytosolic subunits (p47phox, p67phox, p40phox, and Rac2). There appear to be at least three isoforms of NADPH oxidase expressed in the vascular wall. e. Nitric oxide synthase: NO synthases (NOS) are a family of enzymes that convert the amino acid L-arginine to L-citrulline and NO. All NOS isoforms are homodimeric enzymes that require the same substrate (L-arginine), cosubstrates (molecular oxygen, NADPH) and cofactors such as FMN, FAD, tetrahydrobiopterin (BH4) and hem group.

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Three main isoenzymes exist in mammals that are regulated by distinct genes: a constitutive neuronal NOS (nNOS or NOS I), an endotoxin- and cytokine-inducible NOS (iNOS or NOS II) and a constitutive endothelial NOS (eNOS or NOS III). Neuronal NOS performs an important role in intracellular communication. Inducible NOS uses NO to induce oxidative stress on pathogens. Endothelial NOS plays a major role in the regulation of vascular function. For instance, eNOS synthesizes NO by a two-step oxidation of the amino acid L-arginine thereby leading to activation of guanylyl cyclase (sGC). The resulting second messenger cGMP in turn activates the cGMP-dependent kinase, which leads to decrease in intracellular Ca+2 concentrations thereby causing vasorelaxation. However it has become clear, from studies with the purified enzyme, that eNOS may become uncoupled in the absence of the NOS substrate L-arginine or the cofactor tetrahydrobiopterin (BH4). Uncoupled state results in the production of O2•− rather than NO [27, 28]. The key mechanisms causing eNOS uncoupling are attributed to a decrease in intracellular BH4 levels caused either by ONOO--induced BH4 oxidation or by decreased activity of the guanosine triphosphate cyclohydrolase I enzyme and the dihydrofolate reductase, both related to BH4 synthesis [29].

f. Mieloperoxidase: The mieloperoxidase enzyme (MPO) produces hypochlorous acid (HOCl) from H2O2 and chloride anion (Cl-) during the neutrophil's respiratory burst. It requires heme as a cofactor. In addition, it oxidizes tyrosine to tyrosyl radical using H2O2 as oxidizing agent [30]. Both HOCl and tyrosyl radical are cytotoxic, and used by the neutrophil to kill bacteria and other pathogens. g. Cytochrome P450: The membrane-bound microsomal monooxygenase is a multienzyme system that generally summarizes as cytochrome P450 (C-P450), as the terminal oxidase and an FAD/FMN-containing NADPH-cytochrome P450 reductase (CPR). The most common reaction catalyzed by the C-P450 is a monooxygenase reaction. This might be, for example, the insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to H2O RH + O2 + 2H+ + 2e– → ROH + H2O One ROS-generating way is given by ferric P450. Once bounded to the substrate, ferric P450 reduces CPR by accepting its first electron, thereby being reduced. Then, this new ferrous hemoprotein binds an oxygen molecule to form oxycomplex, which is further reduced to give peroxycomplex. The input of protons to this intermediate can result in the heterolytic cleavage of the O–O bond, producing H2O and the ‘oxenoid’ complex, the latter of which then inserts the heme-bound activated oxygen atom into the substrate molecule. Finally, the decomposition of this final one-electron-reduced ternary complex results in O2•– release. The second ROS-producing branch is the protonation of the peroxycytochrome P450 with the formation of H2O2 [31].

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2.3. Pathophysiological Conditions In pathophysiological conditions, the main sources of ROS include the mitochondrial respiratory electron transport chain, XO activation through ischemia–reperfusion, the respiratory burst associated with neutrophil activation, and arachidonic acid (AA) metabolism. Activated neutrophils produce O2•– as a cytotoxic agent as part of the respiratory burst via the action of membrane-bound NADPH oxidase on O2. Neutrophils also synthesize NO that can react with O2•– to produce ONOO-, a powerful oxidant, which may decompose to form •OH. Additionally, in ischemia-reperfusion XO catalyzes the formation of uric acid with the co-production of O2•–. The enhanced O2•– released results in the recruitment and activation of neutrophils and their adherence to endothelial cells, which in turn stimulates the formation of XO in the endothelium, with further O2•– production as a positive feedback model pathway. Accordingly, allopurinol, a XO inhibitor, has been demonstrated that blocks the O2•– production in ischemia–reperfusion settings involving organs such as heart [32], liver [33], kidney [34], and small intestine [35].

2.4. Reactive Oxygen Species as Mediators of Cell Damage As mentioned previously, ROS have physiological functions that are essential in cells, such as mitochondrial respiration, prostaglandin production pathways and host defense [36]. Moreover, NO plays an important role in antagonizing the vasoconstrictor effects of Angiotensin II (Ang-II), endothelins and ROS [37]. However, ROS have well known involvement in common-shared pathophysiological models causing cell damage, either directly or through behaving as intermediates in diverse signaling pathways, including DNA damage, protein oxidation and lipid peroxidation resulting, among others, in membrane damage [38]. 2.4.1. DNA Damage Oxidative DNA modifications are frequent in mammalian and have been suggested as important contributory factors to the mechanism in carcinogenesis, diabetes and natural aging. The DNA damages are considered as the most serious ROS-induced cellular modifications as DNA is not synthesized de novo but copied, perpetuating by this way those modifications and hence inducing mutations and genetic instability. The main responsible ROS of DNA damage is •OH, which reacts with all components of the DNA molecule, damaging both purine and pyrimidine bases and the deoxyribose backbone. This is explained by the diffusion-limited •OH ability to add to double bonds of DNA bases, abstracting a hydrogen atom from the methyl group of thymine and each of the five carbon atoms of 2-deoxyribose [39]. Further reactions of base and sugar radicals generate a variety of modified bases and sugars, base-free sites, strand breaks and DNAprotein cross-links.

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In addition, RNS such as ONOO- and •NO have also been implicated in DNA damage [40]. This can be explained by the following mechanisms:

A. Direct Damage to DNA through Reactive Nitrogen Species: •

Endogenous formation of carcinogenic N-nitrosamine molecules:

N-nitrosamines are chemical molecules with known carcinogenic ability, because of their conversion to strong alquilant agents. They are synthesized through the reaction of N2O3 and biogenic amines: RNH2

+

N2O3 → RNHO + NO2–

RNHNO → RNNOH → ROH + N2 Endogenous production of these compounds has been demonstrated in immortalized hepatocytes with the SV 40 apes virus. •

Basis deamination:

Primary amines from N-nitrosamine might generate diazonium ions, which transform to alcohols, as shown in the following reaction: RNN+

+

H2O → ROH + N2 + H+

The presence of these amines in the main structure of DNA nitrogenated bases shows that they can be deaminated by NO via N2O3, thereby generating punctual alterations with mutagenic potential [41, 42]. In vitro experiments have provided evidence suggesting that bases deamination through this mechanism seems to have an aimed mutation pattern to puric bases, even though they can also affect pirimidinic ones. The most common mutations are the guanine to adenine transition and back forward. In addition, generation of modified bases, such as oxanin derived from guanine, is also a frequent source of unspecific crossing over between DNA and proteins [43, 44]. Subsequently, DNA is affected either by a mechanism causing genomic instability of the molecule, given by crossing over, or through a suicide mechanism of substrate for enzymatic repairing [43]. It has been demonstrated, in vitro, a higher frequency of simple oligonucleotides chains, rather than double ones. This suggests that the mutagenic mechanism occurs when basis are unprotected, likely in replication and transcription, in which double spiral is open [42]. •

Bases oxidation:

In cultured cells, protocols on activated macrophages show oxidative and deamination damage of DNA [17]. Further analysis of the NO final onset revealed that most part of it was

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transformed to ONOO- [45]. The treatment of DNA plasmids with synthetic ONOO- , and its insertion into biological systems for replication and further analysis, confirmed a range of specific mutations, mainly transversions from guanine to timine, and guanine to cytosine [46]. The oxidant power of ONOO- is also enough to directly damage sugar and creates sites with no nitrogenated bases on DNA, as well as oxidizing and modifying bases thus generating hard-reparation class bases [47]. The production of DNA damage through this mechanism also occurs mostly in simple chain DNA.

B. Indirect Modifying DNA Sequence by Reactive Nitrogen Species Some authors have suggested that either deamination, oxidation and DNA chain rupture by RNS requires extremely high concentrations of these species, a situation that would be exceptionally possible in humans. Moreover, in vivo, some antioxidants molecules such as ascorbate and reduced glutathione (GSH) are abundant, thus the RNS possibilities of accumulation at enough concentrations to produce direct DNA damage are extraordinarily low [48]. One of the suggested hypotheses is based on the inhibition of the DNA repairing enzymatic systems, thereby making possible indirect damage. The RNS have a high affinity for the thiol group (-SH) of cysteine [49] and it is believed that those enzymes containing critic cysteine for their activity might be inhibited through RNS. Other nucleophilic groups, such as hydroxyl (-OH) from tyrosine [50] and amine (-NH2) of lysine [23] are also potentially modifiable. All of the mechanisms exposed before contribute to elucidate from diverse points of view the mutagenic effects of NO. While being on the right position, these mutations could result in the inactivation of suppressor tumor genes, and further activation of oncogenes, thus participating in various stages of carcinogenic process. The most evident example of this is given by the protein for p53 gen, which is mutated on nearly 50% of human tumors [51]. Previous researches confirmed in vitro mutations in p53 gene induced by NO and its methylation [52]. Further investigations also verified that there is a significant relation between the RNS activities and the mutations on p53 gene in early staged lung carcinoma [53]. Even though in these studies the functionality of the genetic product was not analyzed, the hypothesis on the role of NO is clear enough to consider that NO, in stress conditions such as inflammation, is able to inactivate p53 gene, therefore to create a favorable environment for tumors emerging and development. 2.4.2. Lipid Peroxidation It is known that ROS attack cellular components involving polyunsaturated fatty acid (PUFA) residues of phospholipids, which are extremely sensitive to oxidation [54]. The overall process of lipid peroxidation consists of three stages: initiation, propagation, and termination [55, 2]. Once formed, peroxyl radicals can be rearranged via a cyclization reaction to endoperoxides, being malondialdehyde (MDA) the final product [56]. The MDA

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is a minor lipid peroxidation product generated by heating of endoperoxides derived from arachidonic acid. The main PUFA in tissue is linoleic acid, five times more abundant than arachidonic acid. Linoleic acid generates only traces of MDA [57], but it is transformed as easily as arachidonic acid to peroxyl radicals. The F2-isoprostanes are useful to demonstrate the occurrence of non-enzymatic lipid peroxidation processes, nevertheless they are only trace products formed through free radicals catalyzed attack on esterified arachidonate, providing a reliable tool to identify population with enhanced rates of lipid peroxidation [58]. Lipid peroxidation involves low-density lipoprotein (LDL) as well as high-density lipoprotein (HDL) oxidation. It is well known that the LDL oxidation is a key process in the pathogenesis of atherosclerosis (chapter 3) [59]. The oxidized cholesterol esters are directly incorporated into lipoproteins and transferred to endothelial cells via the LDL where they induce damage and start the sequence of events leading to atherosclerosis. 2.4.3. Protein Oxidation The side chains of all amino acid residues of proteins are susceptible to oxidation by the action of ROS [60]. The protein carbonyl group is generated by ROS through many different mechanisms and its concentration is a good measure of protein oxidation via oxidative stress. The NO reacts rapidly with O2•– to form the highly toxic ONOO- that is able to nitrosate the cysteine sulfhydryl groups of proteins, to nitrate tyrosine and tryptophan residues of proteins and to oxidize methionine residues to methionine sulfoxide [1]. Oxidation of proteins is associated with a number of age-related diseases and aging [61, 62]. 2.4.4. Other Damage Oxidative damage to the mitochondrial membrane can also occur, resulting in membrane depolarization and the uncoupling of oxidative phosphorylation, with altered cellular respiration [63]. This can ultimately lead to mitochondrial damage, with release of cytochrome c, activation of caspases and apoptosis [64].

2.5. Antioxidant Defense System All forms of life maintain a reducing environment within the cells. The maintenance of this status is achieved possibly through the antioxidant defense system, which is in action to protect cellular homeostasis against harmful ROS produced during normal cellular metabolism, as well as in the pathophysiological states. The antioxidant system is preserved by antioxidant substances that maintain the reduced state by a constant input of metabolic energy. Antioxidant substances are small molecules that can scavenge free radicals by accepting or donating an electron to eliminate the unpaired condition. Typically, this means that the antioxidant molecule becomes a free radical in the process of scavenging a ROS to a more stable and less reactive molecule. In most cases the scavenger molecule provides hydrogen radical that combines with the free radical. Consequently, it is generated a new radical that has an enhanced lifetime compared with the starting one, for instance, due to a conjugated

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system [59]. The extended lifetime of this radical enables it to react with a second radical by formation of a new molecule and thus one scavenger molecule can eliminate two radicals. Antioxidant molecules can be produced endogenously or provided exogenously through diet or antioxidant supplements. The main endogenous antioxidant enzymes are SOD, catalase (CAT), and glutathione peroxidase (GSH-Px). The SOD converts superoxide anion to H2O2, which is a substrate for CAT and GSH-Px. Catalase metabolizes H2O2 to water and oxygen and GSH-Px reduces both H2O2 and organic hydroperoxides when reacting with GSH [65]. Reduced glutathione is present at high concentrations in all mammalian cells, especially in the renal cells, hepatocytes, and erythrocytes [66]. This tripeptide protects protein thiol groups from non-enzymatic oxidation or as a co-substrate of GSH-Px [67]. The endogenous antioxidant defense system is summarized on Table 1. Exogenous antioxidants, such as vitamins E and C, exist at a number of locations namely on the cell membrane, intracellularly and extracellularly. They react with ROS to either remove or inhibit them. The hydrophobic lipid interior of membranes requires a different spectrum of antioxidants. Fat-soluble vitamin E is the most important antioxidant in this environment, which protects against the loss of membrane integrity. Fat-soluble antioxidants are important in preventing membrane polyunsaturated fatty acids (PUFA) from undergoing lipid peroxidation. Glutathione removes already generated radical, if no radicals are present, the PUFA cannot be attacked. Therefore, they shield the membrane rich in PUFA against ROS [68]. In addition, water-soluble antioxidants including vitamin C play a key role in scavenging ROS in the hydrophilic phase. Other small antioxidant molecules are also naturally present in the plasma, such as uric acid and bilirubin. Recently, it was found that fish, fish oils, and some vegetables contain furan fatty acids that are radical scavengers, partly responsible for the beneficial efficiency of a fish diet [69]. Table 1-1. Functional characteristics of the antioxidant enzymes Antioxidant enzyme GSH-Px

Chemical name Glutathione peroxidase

Scavenged oxidant agent H2O2

SOD

Superoxide dismutase

O2•-

CAT

Catalase

H2O2

General characteristics It is the major endogenous antioxidant molecule It catalyzes the conversion of H2O2 and organic peroxides into water or alcohols, respectively. It catalyzes the conversion of O2•− to O2 and to less-reactive species like H2O2. Necessary for the release of biologically active NO. It protects NO from inactivation. It catalyzes the breakdown of H2O2 to water and molecular oxygen.

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3. Reactive Oxygen Species as Factors for Diseases Development In physiological conditions, both enzymatic and non-enzymatic systems preserve the oxidant/antioxidant status. However, these systems are overwhelmed during oxidative stress, which is a metabolic derangement due to an imbalance caused either by an excessive generation of ROS or by a diminished capacity of the antioxidant defense system. In a simplistic manner, it could be considered that diseases are the result of cell functioning disorders that may lead or not into systemic alterations as a chain reaction result. Impairment of cell function might be caused by several factors, typically more than one acting at the same time, enhancing the same pathophysiological pathway or other. On the other hand, it has been discussed the most important conditions involving ROS either as direct cellular damage agents or as mediators implicated in pathophysiological pathways [70, 71]. Reactive oxygen species are thought to contribute to the pathogenesis of a number of seemingly unrelated disorders, including type 2 diabetes, cancer and aging, heart failure, hypertension, preeclampsia and atherosclerosis, among others. All of these pathologies were important causes of morbidity and mortality on the twentieth century, and have been extensively studied over the past few years. Urbanization, aging and globalized lifestyle changes combine to make chronic diseases, while in-between them deleterious effect of ROS seems to be a constant. In order to further explain this, some examples of widely known syndromes will be briefly described as follow:

3.1. Cardiovascular Disease Cardiovascular diseases are the first cause of death in the world. Recent estimations consider that its prevalence will keep on rising for the next decades to come [72], due to an increase in older population and non healthy lifestyle. Reactive oxygen species have a key role in the homeostasis of the vascular wall and there is compelling evidence pointing to ROS as important factors for the development of cardiovascular disease. 3.1.1. Hypertension Hypertension is probably the most prevalent chronic disease in the world. It is also known that its incidence is still arising, especially in emergent countries. However, it seems that this does not apply in developed countries. In U.S.A, for example, the overall prevalence is 29.3%, or 65 million people, and it has not increased significantly since 1999 [73]. It is a silent and harmful condition, as it constitutes an asymptomatic disease on early stages but represents a key risk factor for many other diseases such as heart or brain stroke, cardiac insufficiency or chronic renal failure, among others. Over the past fifty years notable therapeutic advances, particularly pharmacological ones, have been made for the treatment and control of hypertension. Reactive oxygen species are thought to contribute to the pathogenesis of hypertension through an impairment of endothelial cells and through uncoupled eNOS. Chronic oxidative stress causes senescence of

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endothelial cells. This is characterized by a detachment of endothelial cells or part of the endothelial cell membrane. With the persistence of oxidative stress, the capacity of neighboring endothelial cells to repair endothelial injury is limited, and vascular integrity becomes dependent on the incorporation of endothelial progenitor cells, with lower NO synthesizing capacity [74]. This is called endothelial dysfunction, which has been clearly associated with prognosis in patients with heart failure [75], essential hypertension [76], and peripheral artery disease [77]. In most situations where endothelial dysfunction due to increased oxidative stress is encountered, the expression of the eNOS has been shown to be paradoxically increased rather than decreased [78-81]. The mechanisms underlying increased expression of eNOS are likely to be secondary to increased endothelial levels of H2O2, which increases the expression of eNOS at the transcriptional and translational level [82]. An interesting marker of endothelial dysfunction is the asymmetric dimethyl L-arginine (ADMA) that can compete with Larginine for eNOS and therefore reduce eNOS-derived NO production. Increased ADMA levels have been found in patients with risk factors such as chronic smokers, patients with hypercholesterolemia, and in patients with diabetes and renal insufficiency [83]. There is also some evidence that ADMA may even cause eNOS uncoupling, thereby switching eNOS from a NO to O2•− producing enzyme. Several in vivo animal studies have acknowledged that uncoupled eNOS is a significant O2•− source in diverse pathological conditions, including angiotensin II hypertension [84], and chronic congestive heart failure [85]. Additionally, an increased synthesis of O2•− reduces NO bioavailability by inactivation, leading to ONOO- formation. Then, the following ONOO- protonation will break off, liberating the highly peroxidant •OH. Endothelium is affected by this reaction in two different ways: 1) Nitric oxide scavenging impairs its vasodilating activity, leading to permanent high blood pressure and 2) Hydroxyl radical causes damage in endothelial cells perpetuating by this way, resulting in a vicious cycle [85]. 3.1.2. Stroke and Atherosclerosis The relationship between high stroke risk and chronic oxidative stress has been widely documented. This is mainly due to endothelial dysfunction. Nitric oxide has potent antiatherosclerotic properties because once released from endothelial cells, it works in concert with prostacyclin to inhibit platelet aggregation [86]. Nitric oxide blocks the adhesion of neutrophils to endothelial cells and the expression of adhesion molecules. It is interesting to point out that at high concentrations NO also inhibits the proliferation of smooth muscle cells [87]. Therefore, under all conditions where an absolute or relative NO deficit is encountered, the process of atherosclerosis is being initiated or accelerated. In normal conditions, the physiological stimuli for blood vessels to release NO are shear stress and pulsatile stretch. In order to assess the endothelial function, an intra-arterial infusion of acetylcholine (ACh) is used in the clinic. Once infused into the brachial artery, ACh causes a dose-dependent vasodilation. In the coronary artery the inability for a normal vasodilation response or some degree of vasoconstriction entirely depends on the functional integrity of the endothelium. In the presence of cardiovascular risk factors and endothelial dysfunction ACh will cause vasoconstriction due to stimulation of muscarinergic receptors in the media [83].

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3.2. Metabolic Syndrome and Diabetes The metabolic syndrome (MS) includes a variety of cardio-metabolic abnormalities associated with a high risk of developing type 2 diabetes and cardiovascular disease. The pathophysiological basis of MS is a relative insulin deficit, given by a resistance of either peripheral or hepatic insulin action. Chronic oxidative stress impairs insulin action, as was demonstrated in type 2 diabetics. This impairment might be due to several factors, such as membrane fluidity alterations, decreased availability of NO and increased intracellular calcium content [88]. Oxidative stress is strictly influenced by glycometabolic control either in type 1 or type 2 diabetics [89, 90]. In type 2 diabetics, if glycemic control improves the oxidative stress-related parameters, such as thiobarbituric acid reactant substances, if the latter decrease, the same trend seems to occur for the NO2-/NO3- ratio and cGMP content [91]. It has also been demonstrated that insulin treatment nearly corrects the oxidative stress in type 1 diabetics but only improves it in type 2 diabetics [92]. Because the period of insulin treatment and the HbA1c values were similar, the authors suggested the existence of metabolic differences between the two types of diabetes. Even if in diabetes mellitus the postprandial hyperglycemic spikes have been considered to be a critical event in the pathogenesis of micro and macroangiopathic complications [93]. It is obvious that all the metabolic pathways associated with these spikes increase free radical synthesis and worsen the oxidative state. Indeed, in diabetic patients the oxidative stress, physiologically induced by a standard meal, is enhanced. Diabetic patients show, during the postprandial period, an increase in MDA plasma levels and a decrease in sulphydryl groups, α-tocopherol and total radical-trapping antioxidant parameter [94]. The latter observation might be related to the significantly lower levels of urate and ascorbic acid observed in diabetic patients, although dietary intake should be considered when explaining changes in plasma antioxidant levels [95].

3.3. Neurodegenerative Diseases 3.3.1. Alzheimer's Disease A major obstacle in the research for treatment of Alzheimer's disease (AD) is the lack of knowledge about the etiology and pathogenesis of selective neuron death. In recent years, considerable data have been accumulated indicating that the brain in AD is under increased oxidative stress and this may have a role in the pathogenesis of neuron degeneration and death in this disorder. The following evidence strongly supports the role of oxidative stress in AD:

1. Increased brain Fe, Al, and Hg found in AD is capable of stimulating radical species. 2. Increased lipid peroxidation and decreased PUFA in the AD neurons, with a concomitant increase of 4-hydroxynonenal, an aldehyde product of lipid peroxidation, is found in AD ventricular fluid. 3. Increased protein and DNA oxidation is a characteristic of an AD patient brain.

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4. Diminished energy metabolism and decreased cytochrome c oxidase levels are observed in AD brains. 5. Advanced glycation end products (AGE), malondialdehyde, carbonyls, peroxynitrite, heme oxygenase-1 and SOD-1 are found in neurofibrillary tangles and AGE, heme oxygenase-1, SOD-1 in senile plaques of AD brains. 6. A number of studies show that amyloid beta peptide is capable of generating free radicals. Supporting indirect evidence comes from a variety of in vitro studies showing that free radicals are capable of mediating neuron degeneration and death. Overall, these studies indicate that free radicals are possibly involved in the pathogenesis of neuron death in AD. Because tissue injury itself can induce oxidative stress, it is not known whether ROS generation is a primary or secondary event. Even if free radical generation is secondary to other initiating causes, they are deleterious and part of a cascade of events that can lead to neuron death, suggesting that therapeutic efforts aimed to the removal of ROS or prevention of their formation may be beneficial in ameliorating the development of AD [96]. 3.3.2. Parkinson’s Disease Parkinson’s disease (PD) is the most common neurodegenerative disease, and the majority of PD cases involve the sporadic form of PD. Although the etiology of the sporadic form is unknown, mitochondrial dysfunction and oxidative stress are considered to play a prominent role in its pathogenesis. The discovery of the genes that are linked to a rare familial form of PD has provided crucial insights into the molecular mechanisms involved in the pathogenesis of PD. Recent findings implicate mitochondrial dysfunction associated with oxidative damage and abnormal protein accumulation (ubiquitin/proteosome pathway) as the key molecular mechanisms compromising dopaminergic neurons in familial PD. Mutations in Parkin, PTEN-induced kinase 1 (PINK1) and DJ-1 are found in autosomal recessive forms of PD. Recent studies on these genes suggest the central importance of mitochondrial dysfunction and oxidative stress in PD. The above mentioned 3 proteins may be biologically related to each other and may protect the mitochondria against oxidative stress and other harmful stimulations. In particular, Parkin seems to be the most important factor that improves the mitochondrial dysfunction [97].

3.4. Pre-Eclampsia Pre-eclampsia is a human pregnancy-specific disorder that adversely affects the mother, by vascular dysfunction, and the fetus through intrauterine growth restriction. The incidence of preeclampsia is about 5% of all pregnancies, and it constitutes the leading cause of maternal mortality in developed countries. Preeclampsia is characterized by vasospasm, increased peripheral vascular resistance, and thus reduced organ perfusion [98].

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The etiology and pathogenesis of this pregnancy syndrome remains poorly understood. There is substantial evidence to suggest that the diverse manifestations of preeclampsia, including altered vascular reactivity, vasospasm, and discrete pathology in many organ systems, are derived from pathologic changes within the maternal vascular endothelium. The key event leading to the clinical manifestations of preeclampsia is endothelial cell dysfunction likely caused, among other factors, by an increase in ROS and RNS concentration [99]. Defective spiral arteries remodeling causes reduced uteroplacental perfusion, which primarily may contribute to intrauterine growth restriction. In addition, maternal dyslipidemia or a primary or secondary decrease of antioxidants makes preeclampsia increasingly to develop. However, the mechanisms involved in induction of endothelial cell dysfunction remain to be determined. There is evidence for increased nitrotyrosine formation in the preeclampsia placenta due to ONOO- production, perhaps arising from local NO production. In addition, increased xanthine oxidase generation of O2·- and either regionally decreased or inadequate SOD could also be involved. Then, oxidative stress may be the point at which multiple factors converge resulting in endothelial cell dysfunction and the consequent clinical manifestations of preeclampsia [100].

3.5. Glaucoma Increasing evidence indicates that ROS play a key role in the pathogenesis of primary open angle glaucoma (POAG), the main cause of irreversible blindness worldwide. Oxidative DNA damage is significantly increased in the ocular epithelium regulating aqueous humor outflow. This is demonstrated in the appearance of the trabecular meshwork (TM) of glaucomatous patients compared to controls. The pathogenic role of ROS in glaucoma is supported by various experimental findings, including the followings: a b c

Resistance to aqueous humor outflow is increased by H2O2, which induces TM degeneration. Trabecular meshwork possesses remarkable antioxidant activities, mainly related to SOD, CAT and glutathione pathways that are altered in glaucoma patients. In glaucoma patients, intraocular pressure and severity of visual-field defects increase in parallel to the amount of oxidative DNA damage affecting TM.

Vascular alterations, which are often associated with glaucoma, could contribute to the generation of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal cells, seems to be involved in the neuronal cell death affecting the optic nerve in POAG. The highlighting of the pathogenic role of ROS in POAG has implications for the prevention of this disease. The latter is supported by the growing number of studies using genetic analyses to identify susceptible individuals, and of clinical trials testing the efficacy of antioxidant drugs for POAG management. [101]

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3.6. Aging and Cancer Aging has drawn attention to an issue that is of particular relevance to the organization of health systems: the increasing frequency of multimorbidity. in the industrialized world, as many as 25% of 65–69 year olds and 50% of 80–84 year olds are affected by two or more chronic health conditions simultaneously [72]. Aging can be defined as a progressive decline in the ability of the organism to resist stress, damage, and disease. Although there are currently over 300 theories to explain the aging phenomenon, it is still not well understood why organisms age and the reason why the aging process can vary so much in speed and quality from individual to individual. The oxidative stress hypothesis is one of the prevailing theories of aging. This theory states that ROS produced during cellular respiration damage cell lipids, proteins and DNA, accelerate the aging process and increase the risk of disease. It has been hypothesized that the production of free radicals is dependent upon resting metabolic rate and this may have an impact on the aging process [102]. Damage of DNA and thus cancer, is pathophysiologically related with ROS, as mentioned in diverse parts of this chapter.

3.7. Renal Damage Considerable experimental evidence supports the view that ROS could play a key role in the pathophysiological processes of renal diseases [103], including chronic renal failure, hemodialysis, rhabdomyolysis-induced acute renal failure, renal fibrosis, glomerulosclerosis, kidney stones formation, and hyperlipidemia, among others. The abundance of PUFA makes the kidney an organ particularly vulnerable to ROS attack [104]. The involvement of ROS in the mechanism of renal damage is supported by two lines of experimental evidence: (i) detection of products of oxidant injury in renal tissue or urine, and (ii) experimental demonstration of a protective effect of metabolic inhibitors of ROS [105], such as antioxidant vitamins or antioxidant compounds proper of the Mediterranean diet. It is thought that oxidative stress up-regulates the expression of adhesion molecules, chemoattractant compounds and inflammatory cytokines [106]. The glomerulus is considerably more sensitive to oxidative injuries than other nephron segments. Oxidative stress may alter the structure and function of the glomerulus because of the effects of ROS on mesangial and endothelial cells [107]. Reactive oxygen species are increasingly believed to be important intracellular signaling molecules in mitogenic pathways involved in the pathogenesis of glomerulonephritis. In mesangioproliferative glomerulonephritis, the increase of ROS is thought to be produced by a pronounced dysregulation of pro-oxidative and anti-oxidative enzymes leading to a net increase in glomerular ROS levels [108].

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4. Conclusions and Perspectives As a final conclusion, it should be mentioned that oxidative stress plays a major role in cellular damage, and it is involved in several mechanisms leading to a variety of unrelated diseases. The data exposed in this chapter supports the view that the different interaction between oxidant and antioxidant agents, and thus ROS and/or RNS generation, is somehow implicated in a wide range of disease development. Treatments that enhance the antioxidant system are expected to effectively ameliorate cell damage. It should be expected an amelioration on cell damage by means of enhancing the antioxidant system. The following chapters will discuss the possible mechanisms by which ROS and/or RNS are linked with the pathogenesis and the development of well known and highly prevalent human diseases and the studies performed to counteract ROS deleterious effects, by means of a reinforcement of the antioxidant defense system.

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[12] McCord JM. Free radicals and inflammation: protection of synovial fluid by superoxide dismutase. Science. 1974;185:529-31. [13] Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-6. [14] Ignarro LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ. Res. 1987;61:866-79. [15] Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium derived relaxing factor. Nature. 1987;327: 524-526. [16] Granger DN et al. Superoxide radicals in feline intestinal ischemia. Gastroenterol. 1981;81: 22-29. [17] Tamir S, Tannenbaum SR. The role of nitric oxide (NO·) in the carcinogenic process. Biochim. Biophys. Acta. 1996;1288:F31-6. [18] Thomas DD, Liu X, Kantrow SP, Lancaster JR. Biological life-time of nitric oxide: implications for the perivascular dynamics of NO an O2. Proc. Natl. Aca. Sci. USA. 2001;98:355-60. [19] Espey MG, Miranda KM, Thomas D, Wink DA. Distinction between nitrosating mechanism within human cells and aqueous solution. J. Biol. Chem. 2001;276:3008591. [20] Rafikova O, Rafikov R, Nudler E. Catalysis of S-nitrosothiols formation by serum albumin: the mechanism and implication in vascular control. Proc. Natl. Aca. Sci. USA. 2002;99:5913-8. [21] Liu X, Miller MJS, Joshi MS, Thomas DD, Lancaster JR. Accelerated reaction of nitric oxide with O2 within the hydrofobic interior of biological membranes. Proc. Natl. Aca. Sci. USA. 1998;95:2175-9. [22] Gryglewski RJ, Moncada S, and Palmer RM. Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells. Br. J. Pharmacol. 1986; 87: 685–694. [23] Espey MG, Thomas DD, Miranda KM, Wink DA. Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide. Proc. Natl. Aca. Sci. USA. 2002;99:11127-32. [24] Daiber A, Frein D, Namgaladze D,Ullrich V. Oxidation and nitrosation in the nitrogen monoxide/superoxide system. J. Biol. Chem. 2002;277:11882-8. [25] Stohs S.J,. Bagchi D, Oxidative mechanisms in the toxicity of metal-ions, Free Rad. Biol. Med. 1995;18: 321–336. [26] Liochev S.I,. Fridovich I, The Haber-Weiss cycle - 70 years later: an alternative view, Redox report 2002;7: 55–57. [27] Vasquez–Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, and Pritchard KA, Jr. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc. Natl. Acad. Sci. USA. 1998; 95: 9220–9225. [28] Xia Y and Zweier JL. Direct measurement of nitric oxide generation from nitric oxide synthase. Proc. Natl. Acad. Sci. USA. 1997;94:12705–12710.

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[64] Macdonald J., Galley H.F., Webster N.R. Oxidative stress and gene expression in sepsis. Br. J. Anaesth. 2003;90: 221–232. [65] Andreoli SP: Reactive oxygen molecules, oxidant injury and renal disease. Pediatr. Nephrol. 1991; 5:733. [66] Sehirli AÖ, Sener G, Satiroglu H. Protective effect of N-acetylcysteine on renal ischemia/reperfusion injury in the rat. J. Nephrol. 2003; 16:1. [67] Meister A, Anderson ME: Glutathione. Annu. Rev. Biochem. 1983; 52:711. [68] Spiteller G. Are changes of the cell membrane structure causally involved in the aging process? Ann. N. Y. Acad. Sci. 2002 ;959: 30–44. [69] Spiteller G. The relation of lipid peroxidation processes with atherogenesis: a new theory on atherogenesis. Mol. Nutr. Food Res. 2005;49: 999–1013. [70] Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Lab. Invest. 1982;47: 412-26. [71] Mantle D, Preedy VR. Free radicals as mediators of alcohol toxicity. Adverse Drug React. Toxicol. Rev. 1999;18: 235-52. [72] WHO. The World Health Report 2008: Now more than ever. Ch1: p8. [73] Ong KL, Cheung BM, Man YB, Lau CP, Lam KS. Prevalence, awareness, treatment, and control of hypertension among United States adults 1999-2004. Hypertension. 2007;49:69-75. [74] Deanfield JE, Halcox JP, Rabelink TJ. Endothelial function and dysfunction: testing and clinical relevance. Circulation. 2007;115:1285–95. [75] Heitzer T, Baldus S, von Kodolitsch Y, Rudolph V, and Meinertz T. Systemic endothelial dysfunction as an early predictor of adverse outcome in heart failure. Arterioscler. Thromb. Vasc. Biol. 2005;25: 1174–1179. [76] Perticone F, Ceravolo R, Pujia A, Ventura G, Iacopino S, Scozzafava A, Ferraro A, Chello M, Mastroroberto P, Verdecchia P, and Schillaci G. Prognostic significance of endothelial dysfunction in hypertensive patients. Circulation. 2001 ;104: 191–196. [77] Gokce N, Keaney JF, Jr., Hunter LM, Watkins MT, Nedeljkovic ZS, Menzoian JO, and Vita JA. Predictive value of noninvasively determined endothelial dysfunction for longterm cardiovascular events in patients with peripheral vascular disease. J. Am. Coll. Cardiol. 2003; 41: 1769–1775. [78] Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, and Channon KM. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002;105:1656–1662. [79] Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, and Munzel T. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ. Res. 2001;88: E14–22. [80] Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, and Harrison DG. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001;103: 1282–1288.

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[81] Vaziri ND, Ni Z, and Oveisi F. Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats. Hypertension. 1998; 31: 1248–1254. [82] Drummond GR, Cai H, Davis ME, Ramasamy S, and Harrison DG. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ. Res. 2000;86:347–354. [83] Münzel T, Sinning C, Post F, Warnholtz A, Schulz E. Pathophysiology, diagnosis and prognostic implications of endothelial dysfunction. Ann. Med. 2008;40:180-96. [84] Mollnau H, Wendt M, Szocs K, Lassegue B, Schulz E, Oelze M, Li H, Bodenschatz M, August M, Kleschyov AL, Tsilimingas N, Walter U, Forstermann U, Meinertz T, Griendling K, and Munzel T. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ. Res. 2002;90: E58–65. [85] Mollnau H, Oelze M, August M, Wendt M, Daiber A, Schulz E, Baldus S, Kleschyov AL, Materne A, Wenzel P, Hink U, Nickenig G, Fleming I, and Munzel T. Mechanisms of increased vascular superoxide production in an experimental model of idiopathic dilated cardiomyopathy. Arterioscler. Thromb. Vasc. Biol. 2005;25: 2554–2559. [86] Galle J, Wanner C. Oxidative stress and vascular injury--relevant for atherogenesis in uraemic patients? Nephrol. Dial. Transplant. 1997;12:2480-3. [87] Radomski MW, Palmer RM, Moncada S. The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br. J. Pharmacol. 1987;92: 639–46. [88] Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J. Clin. Invest. 1989; 83:1774–7. [89] Paolisso G, Giugliano D. Oxidative stress and insulin action: is there a relationship? Diabetologia. 1996 ;39:357-63. [90] Ceriello A, Giugliano D, Quatraro A, Dello Russo, Lefèbvre PJ. Metabolic control may influence the increased superoxide generation in diabetic serum. Diab. Med. 1991;8:540-42 [91] Aydin A, Orhan H, Sayal A, Özata M, Sahin G, Isimer A. Oxidative stress and nitric oxide related parameters in type II diabetes mellitus: effects of glycemic control. Clin. Biochem. 2001; 34:65-70 [92] Seghrouchni I, Drai J, Bannier E, Riviere J, Calmard P, Garcia I, Orgiazzi J, Revol A. Oxidative stress parameters in type 1, type 2 and insulin-treated type 2 diabetes mellitus; insulin treatment efficiency. Clin. Chim. Acta. 2002 ;321:89-96. [93] Ceriello A. The emerging role of post-prandial hyperglycaemic spikes in the pathogenesis of diabetic complications. Diabet. Med. 1998;15:188-93. [94] Ceriello A, Lizzio S, Bortolotti N, Russo A, Motz E, Tonutti L, Crescentini A, Taboga C. Meal-generated oxidative stress in type 2 diabetic patients. Diabetes Care. 1998; 21:1529-33. [95] Kuyvenhoven JP, Meinders AE. Oxidative stress and diabetes mellitus. Pathogenesis of long-term complications. Eur. J. Intern. Med. 1999;10:9-19. [96] Markesbery WR. Oxidative stress hypothesis in Alzheimer's disease. Free Radic. Biol. Med. 1997; 23:134-47.

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[97] Mitsui T, Kuroda Y, Kaji R. Parkin and mitochondria. Brain Nerve. 2008; 60:923-9. [98] Hauth JC, Cunningham FG. Preeclampsia-eclampsia. In: Lindheimer MD, Roberts JM, Cunningham FG, Eds. Chesley's Hypertensive Disorders in Pregnancy (2nd ed). Stamford, CT: Appleton & Lange 1999; pp. 169–199. [99] Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: An endothelial cell disorder. Am. J. Obstet. Gynecol. 1990; 163:1365– 1366. [100] Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc. Soc. Exp. Biol. Med. 1999; 222:222-35. [101] Izzotti A, Bagnis A, Saccà SC. The role of oxidative stress in glaucoma. Mutat. Res. 2006; 612:105-14. [102] Frisard M, Ravussin E. Energy metabolism and oxidative stress: impact on the metabolic syndrome and the aging process. Endocrine 2006; 29:27-32. [103] Rodrigo R, Rivera G. Renal damage mediated by oxidative stress: A hypothesis of protective effects of red wine. Free Radical Biology and Medicine. 2002; 33:409–22. [104] Kubo, K.; Saito, M.; Tadocoro, T.; Maekawa, A. Changes in susceptibility of tissues to lipid peroxidation after ingestion of various levels of docosahexanoic acid and vitamin E. Br. J. Nutr. 1997; 78:655–669. [105] Ishikawa, I.; Kiyama, S.; Yoshioka, T. Renal antioxidant enzymes: their regulation and function. Kidney Int. 1994; 45:1–9. [106] Klahr, S. Urinary tract obstruction. Semin. Nephrol. 2001; 21:133-145. [107] Klahr S. Oxygen radicals and renal diseases. Miner Electrolyte Metab. 1997; 23:140-3. [108] Gaertner SA, Janssen U, Ostendorf T, Koch KM, Floege J, Gwinner W. Glomerular oxidative and antioxidative systems in experimental mesangioproliferative glomerulonephritis. J. Am. Soc. Nephrol. 2002 ;13:2930-7.

In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter II

Hypertension Ramón Rodrigo Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile Supported by FONDECYT, grant 1070948

Abstract Reactive oxygen species (ROS) and reactive nitrogen species play a key role in the modulation of the vasomotor system. Thus, ROS are recognized as mediators of the vasoconstriction induced by angiotensin II, endothelin-1 or urotensin-II, among others; while nitric oxide (NO) is a major vasodilator. In physiological conditions, low concentrations of intracellular ROS play an important role in normal redox signaling involved in maintaining vascular function and integrity. In addition, under pathophysiological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. The fact that ROS play a key role in development of hypertension is supported by the findings of increased production of superoxide anion and hydrogen peroxide, reduction of NO synthesis, and a decrease in bioavailability of antioxidants in human hypertension. In both animal models and humans, increased blood pressure has been associated with an excessive endothelial production of ROS (oxidative stress) which may be both a cause and an effect of hypertension. Antioxidants, whether synthesized endogenously or exogenously administered, are reducing agents that neutralize these oxidative compounds before they can cause damage to biomolecules. In the management of hypertension and other cardiovascular diseases, the primary interest was focused on the therapeutic possibilities of antioxidants to target ROS, thus avoiding hypertensive end-organ damage. The use of antioxidant vitamins, such as vitamin E and vitamin C, has gained considerable interest for their role as protecting agents against vascular endothelial damage, in this way contributing to ameliorate chronic diseases, beyond its essential function associated to body deficiencies. However, promising findings from experimental investigations, the results of clinical trials aimed to demonstrate antihypertensive effects of antioxidant supplementation are disappointing. Nevertheless, the methodology used in some of these studies makes them

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Ramón Rodrigo a matter still to be debated. Some studies reported a potential antihypertensive effect, particularly when using association of two or more antioxidants. Even more, antioxidant diets low in fat, have found to be of most significant benefit in hypertensive patients. Taken together data are consistent with the view that while an antioxidant alone has not yet demonstrated its efficacy as a therapeutic antihypertensive agent, the synergistic actions among the various antioxidants appear to be effective to counteract the ROS effect on the vascular wall. These effects could arise from their complex biological actions, from their ability not solely to scavenge ROS, but also to prevent their formation through down regulation of NADPH oxidase and up-regulation of endothelial NO synthase and antioxidant enzymes.

1. Introduction Hypertension is considered to be the most important risk factor in the development of cardiovascular disease worldwide [1]. In recent years, oxidative stress has gained widespread attention as one of the fundamental mechanisms responsible for the development of cardiovascular morbidities. Although reactive oxygen species (ROS) have an important role in the homeostasis of the vascular wall, an excessive ROS contributes to impaired endothelium-dependent dilation by decreasing nitric oxide (NO) bioavailability, a pathophysiological condition that leads to hypertension. Increased ROS may be a risk factor for cardiovascular events such as unstable angina, myocardial infarction and sudden death. The understanding of the biological processes that generate ROS and the intracellular signals elicited by ROS is most relevant to gain insight into the pathogenesis of diseases such as hypertension. An increasing body of evidence suggests that oxidative stress could be a contributing factor to the underlying pathophysiological mechanism of hypertension [2-4]. Thus, increased production of superoxide anion and hydrogen peroxide, reduced NO synthesis, and decreased bioavailability of antioxidants have been demonstrated in experimental and human hypertension. The vasculature is a rich source of ROS, which under pathological conditions play an important role in vascular injury, as well as in hypertensive end-organ damage. Vascular ROS are produced in endothelial, adventitial, and smooth muscular cells, and derived primarily from NADPH oxidase that produces superoxide anion when stimulated by hormones such as angiotensin II (Ang-II), endothelin-1 (ET-1) and urotensin II (U-II), among others. In addition, increased ROS production may be generated by mechanical forces, such as both unidirectional laminar and oscillatory shear stress occurring during elevation of blood pressure. Reactive oxygen species function as intracellular second messengers to increase intracellular calcium concentration, a major determinant of vasoconstriction, thereby contributing to the pathogenesis of hypertension. In addition, induction of other signaling cascades leads to vascular smooth muscle cell growth and migration, expression of proinflammatory mediators, and modification of extracellular matrix. Significantly reduced acetylcholine-mediated vasodilation has been partly attributed to elevated ROS and decreased NO bioavailability [5]. Since the regulation of vasomotor tone is dependent upon a delicate balance between vasoconstrictor and vasodilator forces resulting from the interaction of the

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components of the vascular wall and the blood, and both of them can be altered by oxidative stress, the cellular events triggered by ROS significantly contribute to the mechanism of hypertension. These findings have stimulated the interest on antihypertensive therapies targeted against free radicals by decreasing ROS generation and/or by increasing NO bioavailability. Accordingly, antioxidants may be useful in minimizing vascular injury and thereby prevent hypertensive end-organ damage. The next sections of this chapter present the available information pointing to a role of oxidative stress in the mechanism of production of high blood pressure, as well as data related with the use of antioxidants in the prevention or treatment of this pathology.

2. Pathophysiology of Hypertension The role of oxidative stress in the pathophysiology of hypertension will be analyzed on the basis of the interaction of the vascular wall components and effects of vasoactive hormones and factors, in settings altering the vascular homeostasis.

2.1. Role of the Vascular Wall Components The integrity of the vascular wall, composed by endothelium, smooth muscular cells and adventitia, is critical for the maintenance of vascular homeostasis, including the modulation of blood pressure. The regulation of vascular tone may be impaired by changes affecting the interaction between these vascular cells. A description of the physiological role of the vascular wall components is given below. 2.1.1. Endothelium The vascular endothelium is formed by a monolayer of cells that separates the blood from the interstitial compartment and the vascular smooth muscle. It is an autonomous organ that serves not just as a barrier of the transvascular diffusion but is the largest endocrine organ in the body. Endothelial cells adhere to one another through junctional structures formed by transmembrane adhesive proteins that are responsible for homophilic cell-to-cell adhesion. Adherent junctions and tight junctions are the main types of junction. Another kind of junction, the gap junction, allows cells to communicate with each other. The endothelium senses mechanical stimuli, such as pressure and shear stress, and hormonal stimuli, such as vasoactive substances. In response, it releases agents that regulate vasomotor function, trigger inflammatory processes, and affect hemostasis. From the physiological viewpoint, the endothelium is characterized by a wide range of important homeostatic functions. It participates in the control of blood coagulation and fibrinolysis, platelet and leukocyte interactions with the vessel wall, regulation of vascular tone and of blood pressure. Many crucial vasoactive endogenous compounds are produced by the endothelial cells to control the functions of vascular smooth muscle cells and of circulating blood cells. These complex systems determine a fine equilibrium which regulates the vascular tone. Impairments in endothelium-dependent vasodilation lead to the so called endothelial dysfunction. The

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endothelium modulates the balance between opposing mechanisms that are vasodilatation/vasoconstriction, pro-coagulant/antithrombotic, cell proliferation/apoptosis. This dynamic tissue layer constitutes a source and/or target of multiple growth factors and vasoactive mediators involved in regulating the physical and biochemical properties of the systemic vessels, as well as vascular contractility and cell growth. There is no doubt that endothelium plays a regulatory and protective role by generating vasorelaxing substances. However, under pathophysiological processes and circumstances, endothelium-derived vasoconstricting factors can dominate and contribute to deleterious effects. Under some pathophysiological circumstances, e.g. in atherosclerosis or hypertension, endothelium derived vasoconstricting factors can be released and contribute to the paradoxical vasoconstrictor effects. Apart from the peptides ET-1, Ang-II and U-II, other endotheliumderived vasoconstricting agents such as superoxide anions, vasoconstrictor prostaglandins, and thromboxane A2 have been postulated. Indeed, the vascular reactions are the result of a complex interaction of many vasoactive pathways since there are numerous interactions between some of the vasoactive agents released from the endothelium. In normal conditions many factors that stimulate ET-1 synthesis, (e.g. thrombin, Ang-II), also cause the release of vasodilators such as PGI2 and/or NO, which oppose the vasoconstricting action of ET-1. Enhanced production of ET-1 also stimulates mitogenic activity on smooth muscle cells while NO and PGI2 inhibit this proliferative effect [6]. 2.1.2. Vascular Smooth Muscle Cells Functional integrity of vascular smooth muscle cells (VSMC) is essential for good performance of the vasculature. They can modify the luminal diameter, which enables blood vessels to maintain an appropriate blood pressure. In addition, VSMC perform other functions, which become progressively more important during vessel remodeling in physiological conditions such as pregnancy and exercise, or after vascular injury. In these cases, VSMC synthesize large amounts of extracellular matrix components and increase proliferation and migration. Because of these properties, VSMC are fit not only for shortterm regulation of the blood vessel diameter, but also for long-term adaptation, via structural remodeling by changing cell number and connective tissue composition. The main function of vascular smooth muscle tonus is to regulate the caliber of the blood vessels in the body. Excessive vasoconstriction leads to hypertension having physical deleterious effects on the vascular wall, such as tensile stress caused by pressure and the shear stress caused by flow. Tensile stress is the force exerted by blood perpendicular to the vessel wall, whereas shear stress is the dragging frictional force created by blood flow as a tangential pressure on the vessel wall. As a consequence of both effects, a remodeling is induced in vessel wall by changing VSMC characteristics. The effects of shear stress are mediated by the endothelium, which coordinates the response of VSMC to this mechanical stress. In contrast to endothelium-modulated shear stress, stretch acts directly on the VSMC. Mechanical forces appear to enhance the expression of both extracellular matrix and contractile proteins by VSMC in the vessel wall. The molecular mechanisms involved in redox-sensitive cell growth control are poorly understood. Stimulation of cultured VSMC with xanthine/xanthine oxidase increases proliferation, whereas stimulation with hydrogen peroxide causes growth arrest of VSMC. In VSMC, ROS mediate many pathophysiological processes, such as growth,

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migration, apoptosis and secretion of inflammatory cytokines, as well as physiological processes, such as differentiation, by direct and indirect effects at multiple signaling levels. In VSMC it was reported that H2O2 activates phospholipase C through tyrosine phosphorylation and that this activation has a major role in rapid [Ca2+]i mobilization in this type of cells [7]. 2.1.3. Adventitia The tunica external of blood vessels, also known as the tunica adventitia, is a connective tissue coat mainly composed by fibroblasts and collagen. It has been shown that vitamin C is essential for the synthesis of collagen and its deficiency lead to scurvy, an alteration caused by the fact that collagen cannot maintain the blood vessel walls, as collagen serves to anchor them to nearby organs. The importance of the vascular adventitia has been recognized in normal maintenance and homeostasis of vessels as well as in vascular disease. The response of adventitial fibroblast to injury, stretch, cytokines, and hormones can lead to stimulate collagen deposition, differentiation, migration, and proliferation. This response is characterized by increased ROS production by adventitial fibroblast NADPH oxidase, considered an initiator of vascular disease and remodeling. In addition, another source of ROS may be generated by some stimuli, such as Ang-II, causing adventitial accumulation of macrophages enriched in NADPH oxidase, giving rise to a paracrine effect. Therefore, the adventitia can contribute to hypertension by either reducing NO bioavailability or participating in vascular remodeling.

2.2. Role of Vascular Active Hormones and Factors 2.2.1. Acetylcholine Since the discovery in 1980 that acetylcholine (ACh) requires the presence of endothelial cells to elicit vasodilation, the importance of the endothelial cell layer for vascular homeostasis has been increasingly recognized. In vascular vessels with healthy endothelium, ACh induces endothelium-dependent dilation via endothelial muscarinic membrane receptors, which are specific G-protein-coupled receptors, leading to a sequence of Ca2+dependent events that induce the production of endothelial factors, mainly NO by stimulating endothelial NO synthase (eNOS). Nitric oxide then diffuses to underlying VSMC, where it activates guanylyl cyclase to produce cyclic GMP, thus inducing vascular smooth muscle cell relaxation. Administration of exogenous ACh to endothelial cells produces these mediators to cause vasodilation. However, muscarinic cholinergic vasodilation is impaired in the presence of endothelial damage, as occur in coronary atherosclerosis. In this setting ACh may promote smooth muscle-mediated vasoconstriction, a paradoxical vasoconstriction occurring early as well as late in the course of coronary atherosclerosis suggesting that the abnormal vascular response to ACh may represent a defect in endothelial vasodilator function, what may be important in the pathogenesis of coronary vasospasm. In addition, the response to ACh could be reduced by either inhibitors of NOS or cyclooxygenase [8]. It should be emphasized that the diminution in NO bioavailability will lead to significantly reduced ACh-mediated vasodilation [5]. The consequence of an overall increase in ROS is a reduced ability of endothelium to cause vasodilation, thereby accounting for a role of oxidative stress in the

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elevation of blood pressure. Accordingly, as an example seen in diabetes mellitus, a setting involving oxidative stress, the relaxation response of aorta to ACh was found to be significantly decreased compared with control subjects, and the antioxidant resveratrol restored the response to ACh [9]. 2.2.2. Renin-Angiotensin System The renin–angiotensin system (RAS) plays a key role in the development and pathophysiology of hypertension and cardiovascular disease. In hypertension small and large arteries undergo structural, mechanical and functional changes that contribute to vascular complications and increased cardiovascular risk. The major effects are vasoconstriction, endothelial damage and cell growth. Angiotensin-II is the end product of the RAS cascade, a potent vasoactive peptide that can be formed at various sites: in vascular beds rich in converting enzyme. When Ang-II production increases above normal levels, it induces vascular remodeling and endothelial dysfunction in association with increases in levels of blood pressure. As a potent activator of NADPH oxidase, Ang-II through the type 1 Ang-II (AT1) receptor contributes to the production of ROS which participate in a number of different pathologies within the circulatory system [10]. In contrast, NO not solely antagonizes the effects of Ang-II on vascular tone, cell growth, and renal sodium excretion, but also down-regulates the synthesis of angiotensin converting enzyme (ACE) and AT1 receptors. Thus, inhibition of NO synthesis with Nω-nitro-L-arginine methyl ester (L-NAME) induces both increase in blood pressure and heart hypertrophy. The development of oxidative stress may enhance this effect of Ang-II, supported by the finding that Ang-II–dependent hypertension is particularly sensitive to NADPH oxidase–derived ROS. In addition, the repeated administration of Ang-II leads to up-regulation of NADPH oxidase activity. In rats and mice made hypertensive by Ang-II infusion, expression of NADPH oxidase subunits (Nox1, Nox2, Nox4, p22phox), oxidase activity, and generation of ROS are all increased [11, 12]. To investigate the role of NADPH oxidase–derived ROS production in the pathogenesis of Ang-II–sensitive hypertension, various mouse models with altered NADPH oxidase subunit expression have been studied. In p47phox knockout mice and in gp91phox (Nox2) knockout mice, Ang-II infusion fails to induce hypertension, and these animals do not show a significant increase in superoxide production, vascular hypertrophy, and endothelial dysfunction observed in Ang-II–infused wild-type mice [13, 14]. Studies in Nox1-deficient mice demonstrated that vascular superoxide production is reduced, and blood pressure elevation is blunted, in response to Ang-II [15, 16], whereas in transgenic mice in which Nox1 is over expressed in the vascular wall, Ang-II–mediated vascular hypertrophy and blood pressure elevation are enhanced [17]. Numerous signaling pathways activated in response to Ang-II and ET-1 are mediated through the increased level of oxidative stress, which seems to be in causal relation to a number of cardiovascular disturbances including hypertension. On the other hand, ACE inhibition up-regulates eNOS expression. Captopril and enalapril prevented blood pressure rise in young spontaneously hypertensive rats. Captopril, probably due to the antioxidant role of its thiol group, had more effective hypotensive effect than enalapril [18], further supporting the role of oxidative stress in the mechanism of hypertension. In Ang-II–infused mice treated with siRNA targeted to renal p22phox, renal NADPH oxidase activity was blunted, ROS formation was reduced, and blood pressure

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elevation was attenuated, suggesting that p22phox is required for Ang-II–induced oxidative stress and hypertension [19]. Treatment with apocynin or diphenylene iodinium, two pharmacological inhibitors of NADPH oxidase, reduced vascular superoxide production, prevented cardiovascular remodeling, and attenuated the development of hypertension in Ang-II–treated mice [11, 20]. In the vasculature, the endothelial as well as adventitial NADPH oxidase is composed of gp91phox (Nox2) and p22phox, as well as p47phox and p67phox and the G protein Rac1. In vitro studies in VSMC support Ang-II–stimulated Nox1 expression in a protein kinase C (PKC)-dependent fashion [21]. Use of PKC inhibitor GF109203X efficiently inhibited PKC activity, decreased Nox1 basal expression, and abolished Ang-II-induced up-regulation of Nox1 expression. The use of anti-sense Nox1 mRNA in rats completely inhibited Ang-II-induced superoxide production, supporting a role for Nox1 in redox signaling in VSMC. Thus, increased expression of both the endothelial and smooth muscle gp91phox homologues in all the layers of the vessel participate in the superoxide production, which occurs in a PKC dependent fashion [22]. The occurrence of oxidative stress uncouples eNOS, leading to further enhancement in superoxide production. The ability of Ang-II to induce endothelial dysfunction is also due to its ability to downregulate the downstream target of NO soluble guanylyl cyclase, thereby leading to impaired NO/cGMP signaling. Recent studies have demonstrated that autoantibodies against Ang-II type 1 receptor are present in women with preeclampsia. These autoantibodies isolated from the sera of preeclamptic patients behave as Ang-II agonists inducing vasoconstriction in a concentration-dependent fashion. The agonistic effect was completely blocked by losartan, an AT1-receptor antagonist [23]. In addition, the agonistic autoantibodies induce signaling in vascular cells including activating protein-1 and nuclear factor kappa B (NF-κB) activation that results in ROS generation [24]. 2.2.3. Endothelin-1 Endothelins are potent 21 amino acid vasoconstrictor isopeptides produced in different vascular tissues, including vascular endothelium. Endothelin-1 is the main endothelin generated by the endothelium and probably the most important in the cardiovascular system. When ET-1 is administered in large concentrations, it behaves as a potent vasoconstrictor capable of exerting an array of physiological effects, including the potential to alter arterial pressure and circulatory function. Endothelin-1 mediates its effects through two membrane G-protein coupled receptors, ETA and ETB, which exhibit a wide tissue distribution including the endothelial cells, VSMC and adventitial fibroblasts [25]. Endothelin-1 acts through ETA, present only on smooth muscle cells and having mitogenic properties and also mediating contractions. The ETB receptor is located both on smooth muscle cells, where they evoke contractions, and on endothelial cells, inducing relaxation. In the peripheral vasculature, ETA receptors are expressed primarily on the surface membrane of VSMC where they mediate, in large part, the potent and characteristically sustained vasoconstrictor response associated with administration of exogenous ET-1 peptides [26]. Administration of exogenous ET-1 to an intact normotensive animal produces a classic, transient hypotension and vasodilation that is mediated via ETB receptors through enhanced generation of NO and prostaglandin-related substances, a response that precedes ETA-mediated vasoconstriction [27]. In the vasculature, the proendothelin may be released from the non-luminal surface of the endothelial cells and

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converted extracellularly to mature ET-1 by membrane-bound endothelin-converting enzymes, which are neutral metalloproteinases. Endothelin-1 does not appear to be stored in endothelial cells, but is rather synthesized de novo in response to several substances (thrombin, Ang-II, cytokines) or physical stimuli (shear stress, hypoxia). Endothelin-1 is a potent vasoconstricting agent with long lasting effects. In normal conditions there are numerous interactions between some of the vasoactive agents released from the endothelium. Many factors that normally stimulate ET-1 synthesis, (e.g. thrombin, Ang-II) also cause the release of vasodilators such as PGI2 and/or NO, which oppose the vasoconstricting action of ET-1. On smooth muscles cells ET-1 also stimulates mitogenic activity, while NO and PGI2 inhibit this proliferative effect. It should be mentioned that ETB receptors involved in the pressor responses triggered by ET-1 are importantly involved in the plasma clearance of the endogenous peptide. In addition, the endothelial ETB receptor acts as an important modulator of ETA receptor-mediated pressor effects [28]. Endothelin-1 stimulates ROS production through the activation of NADPH oxidase, xanthine oxidase, lipoxygenase, uncoupled NO synthase, and mitochondrial respiratory chain enzymes. With respect to arterial hypertension development, NADPH oxidase seems to be the main enzyme responsible for superoxide production [3]. It was reported that essential hypertension is characterized by increased ET-1 vasoconstrictor tone, an effect that seems to be dependent on decreased endothelial ETBmediated NO production attributable to the impaired NO bioavailability. In such conditions endothelial ETB-induced vasodilation no longer compensates for the direct classical ET-1 vasoconstrictor effect mediated by smooth muscle cell ETA and ETB receptors [29]. Overexpression of human ET-1 in mice also induces vascular remodeling and impairs endothelial function, via activation of NADPH oxidase [30]. Infusion of ET-1 increases NADPH oxidase-dependent superoxide production; however, preventing this increase in ROS generation does not inhibit development of hypertension in these animals [31]. 2.2.4. Urotensin-II Human urotensin-II (U-II) is a potent vasoactive peptide, indeed the most potent vasoconstrictor identified. Urotensin-II is a peptide composed of 11 amino acid residues with a structure similar to somatostatin that was firstly isolated from a fish. Subsequently, human U-II and its receptor were identified. In rat thoracic aorta U-II triggers powerful vasoconstrictor activity, with effects on pulmonary artery smooth muscle cells [32]. This action is brought about via activation of a Gq/11-protein coupled receptor (UT receptor). Urotensin-II activation of the UT receptor increases inositol phosphate turnover and intracellular Ca2+concentration. However, the constrictor response to U-II appears to be variable and highly dependent on the vascular bed examined. Vasoconstriction is not its only effect; U-II and its receptor have been demonstrated in the central nervous system, where UII induces a cardiovascular, behavioral, motor and endocrine response and in the kidney, where it seems to influence renal hemodynamics but also salt and water excretion, in rat pancreas where it inhibits insulin secretion, in the heart where it seems to play a role in cardiac hypertrophy and fibrosis. In humans high plasma or urine levels of U-II have been described in some pathologic conditions. U-II has also been shown to act as a potent vasodilator in some isolated vessels; for example, human small pulmonary and abdominal arteries [33]. In addition to these vascular

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actions, U-II is a positive inotrope in human right atrial trabeculae and also exhibits arrythmogenic activity [34]. Human U-II and its UT receptor display greatest expression in the peripheral vasculature, heart, and kidney [35], although both are found in other tissues, notably the central nervous system [36]. It also appears that it plays a relatively minor role in health, as shown by knockout studies in mice and infusion studies in humans. The role of UII in disease is not well elucidated. Urotensin-II is expressed in endothelial cells, macrophages, macrophage-derived foam cells, and myointimal and medial VSMC of atherosclerotic human coronary arteries. UT receptors are present in VSMC of human coronary arteries, the thoracic aorta and cardiac myocytes. Lymphocytes are the most active producers of U-II, whereas monocytes and macrophages are the major cell types expressing UT receptors. The recent detection of this potent vasoconstrictor in human tissue, and the identification of its receptor in the spinal cord, heart lungs, blood vessels, and brain, have made U-II a major focus of current clinical research and a potential target for future human pharmacotherapy. 2.2.5. Norepinephrine Vascular smooth muscle is innervated primarily by the sympathetic nervous system through adrenergic receptors (adrenoceptors), which are G protein–coupled receptors. Three types of adrenoceptors are present within VSMC: α1, α2 and β2. The main endogenous agonist of these cell receptors is norepinephrine (NE). Norepinephrine stimulates VSMC proliferation through α1-adrenergic receptors via the activation of the Ras/mitogen activated protein kinase (MAPK) pathway, it also stimulates phospholipase D activity in VSMC, an enzyme that catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and choline and whose activation by neurotransmitters, hormones, or growth factors has been implicated in a wide range of cellular responses, including cellular trafficking, inflammatory and immune response, mitogenesis, cellular differentiation, and apoptosis. In addition, overexpression of iNOS increases blood pressure via central activation of the sympathetic nervous system, which is mediated by an increase in oxidative stress. [37]. In turn, Ang-II enhances sympathetic nervous system activity centrally and peripherally, but the exact mechanisms of this activation are not well established. 2.2.6. Nitric Oxide The primary role of the endothelial cells is the modulation of the vascular tone, by producing vasodilator and vasoconstrictor factors. The endothelial cell-derived relaxing factor (EDRF), which was originally described by Furchgott and Zawadzki [38], has been identified as NO and is now known to play an important role as a key paracrine regulator of vascular tone. Nitric oxide has been found to play many diverse physiological roles ranging from a neurotransmitter, a vasodilator to a cytotoxic agent. Physiologically, NO inhibits leukocyte–endothelial cell adhesion, VSMC proliferation and migration, and platelet aggregation to maintain the health of the vascular endothelium. Therefore it has many beneficial effects, including inhibiting thrombosis, inhibiting inflammation, promoting survival of endothelial cells, and inhibiting recruitment of macrophages to the vessel wall. The decrease in bioavailable NO in the vasculature reduces vasodilatory capacity and contributes to hypertension. The enzyme that catalyzes the formation of NO from oxygen and

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arginine is NO synthase (NOS), a very complex enzyme containing several cofactors and a heme group which is part of the catalytic site. Indeed, there is a family of NOS, flavo-heme enzymes that catalyze a stepwise oxidation of L-arginine to form NO and L-citrulline. The NOS isoforms differ with respect to the main mode of regulation, the tissue expression pattern and the average amount of NO produced [39]. Endothelial NOS (eNOS), expressed in endothelial cells, is the predominant NOS isoform in the vessel wall. Receptor-mediated agonist stimulation (e.g. bradykinin, acetylcholine, thrombin, histamine) leads to rapid enzyme activation by depalmitoylation, binding to calmodulin/calcium, displacement of caveolin and release from the plasma membrane [40]. In addition, shear stress is also an important modulator of eNOS activity. Endothelial NOS activity is also regulated by allosteric modulators [41]. Nitric oxide activates guanylyl cyclase by binding to the heme moiety of this enzyme. Guanylyl cyclase catalyzes the conversion of guanosine triphosphate (GTP) to cGMP, which in turn activates cGMP-dependent protein kinase. Except the vasorelaxing and antiproliferative properties per se, NO plays an important role in antagonizing the effects of Ang-II, endothelins and ROS. It was shown previously that ACE inhibition up-regulates eNOS expression. The mechanism of this up-regulation is still unclear. However, it is conceivable that ACE inhibitor-induced accumulation of endogenous kinins mediates this effect [42]. All NOS isoforms are homodimeric enzymes that require the same substrate (L-arginine), cosubstrates (molecular oxygen, NADPH) and cofactors such as FMN, FAD, tetrahydrobiopterin, or heme. Tetrahydrobiopterin (BH4) is bound tightly within NOS and this enables it to remain bound in NOS through multiple catalytic turnovers; it reduces the ferric heme-superoxy intermediate that forms during oxygen activation, and becomes an enzyme-bound BH4 radical in the process [43]. Nitric oxide is released by the endothelium and is a gas that bubbles from the endothelial cell to the VSMC. Nitric oxide diffuses to the adjacent smooth muscle where it interacts with different receptor molecules, of which the soluble guanylyl cyclase (sGC) is the best characterized and presumably most important one with regard to control of vessel tone and smooth muscle proliferation. Activation by NO requires sGC heme-iron to be in the ferrous (II) state. Upon NO binding, cGMP formation will increase substantially. Cyclic GMP in turn activates the cGMPdependent kinase I which in turn will increase the open probability of Ca2+-activated K+(BK)channels, thereby inducing a hyperpolarization of the VSMC and inhibition of agonistinduced Ca2+ influx. During a relatively short time period, our knowledge on the role of endothelium and NO in cardiovascular diseases has tremendously increased. It is accepted that the normal reduction of NO plays a crucial role in the maintenance of the physiologic conditions within the cardiovascular system. L-arginine, a substrate for eNOS, seems to be promising in preserving NO formation. However, L-arginine failed to prevent blood pressure increase and left ventricle remodeling due to chronic treatment with L-NAME, an inhibitor of eNOS [44]. Some other effects of L-NAME, besides blood pressure increase and NO deficiency, could participate in this lack of L-arginine protection. It has been demonstrated that L-NAME inhibits L-arginine transport to the caveolae containing NOS [45]. Moreover, L-NAME increased the activity of NF-κB, which may participate in cardiovascular remodeling independently of the blood pressure increase [46]. The angiotensin converting enzyme inhibitor captopril completely prevented NO-deficient hypertension, yet without improving NOS activity. It was suggested that both inhibition of Ang-II formation and

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enhanced production of PGI2 caused by increased bradykinin level may be responsible for observed protective effect of captopril. Thiols protect NO from oxidation by scavenging oxygen-free radicals and by forming nitrosothiols, both effects prolonging NO half-life and duration of NO action [47, 48]. Interestingly, aldosterone receptor blocker spironolactone was also able to prevent degradation of thiol groups and to increase the expression of eNOS protein, two effects associated with blood pressure reduction [49, 50]. It seems that not the absolute NO production but the relative balance between vasodilators and vasoconstrictors is decisive. Nitric oxide is able to reduce generation of ROS by inhibiting association of NADPH oxidase subunits. The balance between NO and Ang-II in the vasomotor centers seems to play important role in the regulation of the sympathetic tone. Reduced NO levels can be attributed to oxidative stress that is related to elevated levels of ROS, such as superoxide and hydrogen peroxide, together with peroxynitrite. Elevated NADPH oxidase expression and activity leads to high superoxide levels. Superoxide combines with NO to form peroxynitrite that oxidizes BH4 and destabilizes eNOS to produce more superoxide [51, 52], thus further enhancing the development of oxidative stress (see below). Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and also occurs within the smooth muscle cell layer. Increased superoxide production has important consequences with respect to signaling by the sGC and the cGMP-dependent kinase I, which activity and expression is regulated in a redox-sensitive fashion [53]. 2.2.7. Prostaglandins Prostacyclin (PGI2), another endothelium-dependent vasodilator, relaxes the underlying VSMC through activation of adenylyl cyclase and subsequent generation of cAMP. Constitutively released PGI2 appears to be involved in the regulation of resting vascular tone. Prostacyclin is released in higher amount in response to ligand binding on the cell surface such as thrombin, arachidonic acid, histamine, or serotonin. The endothelium has an important function in maintaining vascular tone, which is mediated in part by the enzymes prostaglandin H2 synthase that uses arachidonic acid as a substrate, forming prostaglandin H2 (PGH2). Prostaglandin H2 is converted to vasoactive molecules, such as PGI2 and thromboxane, via specific synthases (prostacyclin synthase and thromboxane synthase, respectively). Prostaglandin H2 synthase (PGHS) has an inducible isoform (PGHS-2), which is oxidant sensitive through the activation of NF-κB, a response also shown by inducible NOS. The isoform PGHS-2 may mediate vascular dysfunction in conditions characterized by oxidative stress. In addition, enhanced NOS activity in an environment of oxidative stress would result in scavenging of NO by superoxide anion generated by endothelial cells and VSMC, forming the potent pro-oxidant peroxynitrite, thus reducing NO bioavailability as a vasodilator. Peroxynitrite can contribute to the altered vascular reactivity in a variety of conditions in which the clinical manifestations are mediated by oxidative stress. Thus, peroxynitrite inhibits the enzymatic activity of prostacyclin synthase, thereby causing impairment in the PGI2-mediated vasodilation. The mechanism by which peroxynitrite inhibits prostacyclin synthase activity involves the nitration of tyrosine 430 [54, 55]. As nitration of this tyrosine residue disrupts the catalytic activity, it has been postulated that this tyrosine residue is embedded in the heme region and is crucial for electron transfer. The

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physiological and pathophysiological implications of prostacyclin synthase nitration by peroxynitrite remain to be evaluated by appropriate in vivo experiments. 2.2.8. Endothelium Dependent Hyperpolarizing Factor and Leukotrienes The endothelium controls vascular tone not only by releasing NO and PGI2 but also by other pathways causing hyperpolarization of the underlying VSMC. Early experimental evidence suggested that, beside the cyclooxygenase and the NOS pathways, an additional endothelial pathway had to be involved to fully explain endothelium-dependent relaxations. Furthermore, it is of interest to note that the endothelial monolayer behaves as a conductive tissue propagating an electrical signal along the axis of the blood vessel by means of homocellular gap junctions and throughout the vascular wall itself by means of myoendothelial gap junctions. It has been suggested that endothelium-dependent relaxations, independent of the production of NO and PGI2, probably play an important role in cardiovascular physiology in the animal and in the human [56]. Therefore, a yet unidentified endothelium-derived hyperpolarizing endothelial factor (EDHF) associated with the hyperpolarization of the VSMC was suggested [57]. Although the nature of EDHF is still controversial, this additional endothelial pathway, endothelium-dependent hyperpolarization, has been demonstrated in many blood vessels of different species, including humans. This factor is the major contributor to endothelium-dependent dilatations induced by agonists such as ACh and bradykinin in small arteries [58]. Nevertheless, the EDHF cannot be defined as a single factor or pathway which accounts for all features of EDHF signaling in different vascular beds and species. This led to the assumption that there are several distinct EDHFs acting alone, in parallel, or even together. Several candidate molecules/mediators have been shown to act as EDHF in different tissues and species. These include K+, cytochrome P450 leukotriene metabolites (epoxyeicosatrienoic acids), lipoxygenase products, NO itself, reactive oxygen species (H2O2), cyclic adenosine monophosphate, C-type natriuretic peptide, among others. Electrical communication between endothelial and smooth muscle cells through gap junctions (myoendothelial gap junctions) has also been suggested to be involved in endothelium-dependent hyperpolarization. Endothelium generates a hyperpolarizing factor, which is suspected to be an arachidonic acid metabolite produced by cytochrome P450. The EDHF contributes to vasodilatation by acting on K+ channels. Under conditions of oxidative stress, a decrease in the bioavailability of NO, as demonstrated in various states associated with endothelial dysfunction, alleviates this intrinsic inhibition so that the activity of the production of the vasodilator epoxyeicosatrienoic acids is increased. As a consequence of this interaction, vascular responsiveness is thought to be at least partially maintained despite the apparent loss of NO.

2.3. Vascular Oxidative Stress Oxidative stress constitutes a unifying mechanism of injury of many types of disease processes, it occurs when there is an imbalance between the generation of ROS and the antioxidant defense systems in the body so that the latter become overwhelmed [59]. By far the dominant situation is increased generation due to alterations in mitochondrial metabolism

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and metabolism of fatty acids and carbohydrates. The ROS family comprises many molecules that have divergent effects on cellular function, such as regulation of cell growth and differentiation, modulation of extracellular matrix production and breakdown, inactivation of NO, and stimulation of many kinases and proinflammatory genes [60-62]. Importantly, many of these actions are associated with pathological changes observed in cardiovascular disease. ROS are produced by all vascular cell types, including endothelial, smooth muscle, and adventitial cells, and can be formed by numerous enzymes. Enzymatic sources of ROS that are important in vascular disease and hypertension are xanthine oxidase, uncoupled NOS, and NADPH oxidase. In pathological conditions, ROS production in vascular tissues, particularly superoxide anions, has been implicated as playing an important role in vascular events such as inflammation, endothelial dysfunction, cell proliferation, migration and activation, extracellular matrix deposition, fibrosis, angiogenesis, all important processes contributing to cardiovascular remodeling in hypertension, atherosclerosis, diabetes, cardiac failure, myocardial ischemia-reperfusion injury, vascular remodeling after angioplasty and ischemic stroke [63-65]. These effects are mediated through redox-sensitive regulation of multiple signaling molecules and second messengers [37, 66, 67]. The elevation of blood pressure has been associated with ROS abundance and frequently also with an impairment of endogenous antioxidant mechanisms [3]. Superoxide, the first ROS formed by one electron reduction of molecular oxygen, and superoxide-derived ROS have multiple pathophysiological actions in the artery wall, including an impairment of endothelium-dependent vasodilation. In agreement with this view, in human hypertension, biomarkers of systemic oxidative stress are elevated [68]. Clinical studies have demonstrated that essential hypertensive patients produce excessive amount of ROS [69, 70], and have abnormal levels of antioxidant status [71], thereby contributing to the accumulating evidence that increased vascular oxidative stress could be involved in the pathogenesis of essential hypertension [4, 72]. Recently, it was demonstrated a strong association between blood pressure and some oxidative stress–related parameters [73]; thus, systolic and diastolic blood pressures of hypertensives were negatively correlated with plasma antioxidant capacity and positively correlated with both plasma and urine 8-isoprostane, a recognized biomarker of oxidative stress in vivo. In the context of oxidative stress in the vasculature it is particularly important to note that increased superoxide reacts extremely rapidly with NO to form peroxynitrite, thereby elevating vascular resistance and promoting vasoconstriction [74]. Formation of peroxynitrite is a pathophysiological process, because NO is an essential endogenous vasodilator. Thus, therapeutic strategies should aim to restore bioavailability of NO, scavenging ROS by antioxidant agents.

2.4. Sources of Reactive Oxygen Species in the Vascular Wall A variety of enzymatic and non-enzymatic sources of ROS exist in blood vessels. Enzymatic sources of ROS include NADPH oxidases located on the cell membrane of polymorphonuclear cells, macrophages and endothelial cells and cytochrome P450-dependent oxygenases. The proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another enzymatic source of both superoxide and H2O2 (and therefore constitutes a

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source of the highly reactive hydroxyl radicals) and has been proposed to mediate deleterious processes in vivo. In addition to NADPH oxidase, the best characterized source of ROS, several other enzymes may contribute to ROS generation, including NO synthase, lipoxygenase, cyclo-oxygenases, xanthine oxidase and cytochrome P450 enzymes. It has been suggested that also mitochondria could be considered a major source of ROS: in situations of metabolic perturbation, increased mitochondrial ROS generation might trigger endothelial dysfunction, possibly contributing to the development of hypertension. However, the use of antioxidants in the clinical setting induced only limited effects on human hypertension or cardiovascular endpoints. 2.4.1. NADPH Oxidase The primary biochemical source of ROS in the vasculature, particularly of superoxide, appears to be the membrane associated nicotinamide dinucleotide (phosphate) (NADH/NADPH) oxidase enzyme complex [75], the major source of superoxide in the vascular wall. This system catalyses the reduction of molecular oxygen by NADPH as electron donor, thus generating superoxide. The function of this enzyme complex is most easily understood in the context of the activated neutrophil, wherein it generates large amounts of toxic superoxide anion and other ROS important in bactericidal function. NADH/NADPH oxidase is also functional in membranes of vascular endothelial and VSMC, and fibroblasts providing a constitutive source of superoxide anion. This enzyme consists of several membrane-bound subunits (gp91, Nox, and p22phox) and cytosolic subunits (p47phox, p67phox, p40phox, and Rac2) [76]. It forms an enzyme complex of VSMC [77]. There appear to be at least three isoforms of NADPH oxidase expressed in the vascular wall (see chapter 1, for more details). Thus, although endothelial cells and adventitial fibroblasts express a gp91phox-containing NADPH oxidase similar to that originally identified in phagocytes, VSMC may rely on novel homologues of gp91phox, namely Nox1 and Nox4, to produce superoxide. Upon assembly of the subunits in the membrane, this enzyme generates a burst of superoxide [78]. NADPH oxidase is up-regulated in hypertension by humoral and mechanical signals, and quantitatively this enzyme makes the largest contribution to ROS production. Genetic and chemical manipulation of NADPH oxidase and of antioxidant enzymes causes predictable changes in oxidative stress and endothelium-dependent function in hypertension. The activity of the enzyme in endothelial as well as VSMC is increased upon stimulation with Ang-II, the most studied stimulus of the vascular NADPH oxidase, although other pressor agents such as ET-1 and U-II are also involved, thereby resulting in increased ROS. This is consistent with the hypothesis that the pathological state of high blood pressure is associated with loss of balance between status of oxidative stress and level of antioxidants. In endothelium, adventitia, and cardiomyocytes, the agonist sensitive NADPH oxidase appears to contain gp91phox. Unlike the phagocytic oxidase, in endothelial cells at least, the gp91phox-based oxidase is constitutively assembled in a perinuclear location associated with the cytoskeleton [79]. It is, however, responsive to stimulation with Ang-II, thrombin, and ET-1, as well as to mechanical forces. Likely the most well known function of NADPH oxidase derived superoxide is inactivation of NO to form peroxynitrite, leading to impaired endothelium dependent vasodilation and uncoupling of eNOS to produce additional superoxide [80]. This scenario, where NADPH oxidase derived superoxide activates other

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enzymes in turn to produce ROS, may be a general mechanism for enhancing free radical formation, because it has been shown that NADPH oxidases are upstream of activation of xanthine oxidase (which also generates ROS) by oscillatory shear stress [81]. Superoxide combines with NO, which is synthesized by eNOS, to form peroxynitrite. In turn, peroxynitrite oxidizes and destabilizes eNOS to produce more superoxide [51, 52]. In the vasculature, NADPH oxidase activation has been strongly associated with hypertension [82]. 2.4.2. Xanthine Oxidase It is an important source for oxygen free radical present in the vascular endothelium [82]. It catalyzes the last two steps of purine metabolism through the sequential hydroxylation of hypoxanthine to yield xanthine and uric acid. During this process oxygen is reduced to superoxide. The enzyme can exist in two forms that differ primarily in their oxidizing substrate specificity. The dehydrogenase form preferentially utilizes NAD+ as an electron acceptor but is also able to donate electrons to molecular oxygen. By proteolytic breakdown as well as thiol oxidation xanthine dehydrogenase from mammalian sources can be converted to the oxidase form that readily donates electrons to molecular oxygen, thereby producing superoxide and hydrogen peroxide, but does not reduce NAD+. Although xanthine oxidase– derived superoxide has been studied mainly in the context of cardiac disease, there is evidence suggesting involvement in vascular dysfunction in hypertension. Spontaneously hypertensive rats demonstrate elevated levels of endothelial xanthine oxidase and increased ROS production, which are associated with increased arteriolar tone [84]. This may be mediated in part through an adrenal pathway, because adrenalectomy reduces xanthine oxidase expression [85]. In addition to effects on the vasculature, xanthine oxidase may play a role in end-organ damage in hypertension. The enzyme inhibitor allopurinol can improve cardiac hypertrophy in spontaneously hypertensive rats but having a minimal impact on blood pressure [86], thereby supporting a role for xanthine oxidase in hypertensive end-organ damage rather than in the development of hypertension per se. It was suggested that this damage may be mediated through direct vascular effects of uric acid [87]. 2.4.3. Uncoupled Endothelial NO Synthase A third potential source of vascular ROS production is eNOS. Endothelial NOS is a cytochrome P450 reductase-like enzyme that requires cofactors including BH4, flavin nucleotides, and NADPH for transfer of electrons to a guanidino nitrogen of L-arginine to form NO. L-arginine and BH4 deficiency are associated with uncoupling of the L-arginineNO pathway resulting in decreased formation of NO, and increased eNOS-mediated generation of superoxide (and peroxynitrite). In agreement with this view, BH4 repletion improves endothelial function in chronic smokers [88], and augments NO bioactivity in hypercholesterolemic humans [89]. The BH4 deficiency, in turn, induces eNOS uncoupling, resulting in the generation of superoxide anions from uncoupled eNOS, which decreases BH4 levels further–a vicious cycle causing endothelial dysfunction [90]. NADPH oxidase is the initial source of ROS leading to BH4 oxidation. In fact, BH4 is highly sensitive to oxidation, e.g., by peroxynitrite, and reduced levels of BH4 promote eNOS uncoupling. In addition, supplementation with BH4 is capable of correcting eNOS dysfunction. Under various pathological conditions, such as substrate/cofactor availability, eNOS activity becomes

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uncoupled, resulting in the production of superoxide rather than NO, thus contributing to the development of hypertension. When NOS is uncoupled, electrons flowing from the reductase domain to the heme are diverted to molecular oxygen instead of to L-arginine, resulting in the formation of superoxide anion [91]. A number of potential mechanisms are responsible for uncoupling of eNOS, although the most consistent evidence exists for BH4 deficiency [92]. This is an essential cofactor in the oxygenase domain and is proposed to have multiple roles in all mammalian NOS isoforms. One of the presently accepted functions of BH4 is to act as a one-electron donor during reductive activation of the oxyferrous complex of the heme. It was reported that eNOS uncoupling is not simply a consequence of BH4 insufficiency, rather it results from a diminished ratio of BH4 vs. its catalytically-incompetent oxidation product, 7,8,-dihydrobiopterin (BH2). The activity of NADPH oxidase is critically important in producing ROS that ultimately oxidize BH4 in blood vessels of hypertensives. The loss of BH4 alters the function of eNOS, resulting in diminished NO production and increased production of ROS from the enzyme. Oral treatment with BH4 or NADPH oxidase deficiency blunts the increase in blood pressure, suggesting that eNOS uncoupling contributes to the progression of hypertension. A similar hypothesis to BH4 deficiency that causes eNOS uncoupling has been also proposed for asymmetric dimethylarginine (ADMA) accumulation. This compound is a naturally occurring amino acid resulting from proteolysis of methylated arginine residues in proteins and it behaves as an endogenous inhibitor of eNOS. It competes with L-arginine to inhibit eNOS for NO production [93]. In the presence of high concentrations of ADMA, eNOS produces superoxide instead of NO. Thus, elevated levels of ADMA and oxidative stress in hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated blood pressure. A recent study demonstrated that hypertensive patients show an improvement of endothelial dysfunction by treatment with nebivolol, a selective 1-adrenergic receptor antagonist. This effect may be related to a diminution of circulating ADMA levels. Although the mechanism by which nebivolol reduces circulating ADMA in these patients remains unclear, it was suggested that the up-regulation of the expression of the enzyme that selectively degrades ADMA (dimethylarginine dimethylaminohydrolase 2) may have a role [94]. 2.4.4. Mitochondria and Microsomes The mitochondrion is a major source and target of ROS. Thus, superoxide formation occurs on the outer mitochondrial membrane, in the matrix and on both sides of the inner mitochondrial membrane. Whilst the superoxide generated in the matrix is eliminated in that compartment, part of the superoxide produced in the intermembrane space may be carried to the cytoplasm via voltage-dependent anion channels [95]. Superoxide is enzymatically converted to H2O2 by superoxide dismutase (SOD), a family of metalloenzymes (for more details see chapter 1). The mitochondrial matrix contains a specific form of SOD, with manganese in the active site (MnSOD), which eliminates the superoxide formed in the matrix or on the inner side of the inner membrane. The expression of MnSOD is further induced by agents that cause oxidative stress, including radiation and hyperoxia, in a process mediated by the oxidative activation of NF-κB. Catalase, a major H2O2 detoxifying enzyme found in peroxisomes, is also present in heart mitochondria. In addition to cytochrome c, other electron carriers appear to have a detoxifying role against ROS. Ubiquinol or coenzyme Q

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has been shown to act as a reducing agent in the elimination of various peroxides in the presence of succinate [96]. Thus, coenzyme Q is a source of superoxide when partially reduced (semiquinone form) and an antioxidant when fully reduced. The inner mitochondrial membrane also contains vitamin E, a powerful antioxidant that interferes with the propagation of free radical-mediated chain reactions. Complex I produces most of the superoxide generated by mammalian mitochondria in vitro during reverse electron transport from succinate to NAD+. Complexes II and IV are not normally significant sites. The high superoxide production from complex I during reverse electron transport is particularly sensitive to mild uncoupling. Therefore mild uncoupling very effectively decreases the high superoxide production that occurs from complex I during reverse electron transport. Superoxide is reactive, but can be converted into hydrogen peroxide by SOD, then to oxygen and water by catalase or glutathione peroxidase. However, superoxide that evades these antioxidant systems (together with the secondary ROS it generates) can damage proteins, lipids and DNA. Although the hydrogen peroxide produced by SOD is relatively unreactive, it can form highly reactive hydroxyl radicals in the presence of ferrous ion via Fenton chemistry and these hydroxyl radicals can initiate lipid peroxidation cascades in membranes. Furthermore, the products of sugar, protein and lipid oxidation can cause secondary damage to proteins. Thus mitochondrially-produced superoxide can be a major source of cellular damage. There are two major side reactions: electrons may leak from the respiratory chain and react inappropriately with oxygen to form superoxide. Glycerol 3-phosphate dehydrogenase produces significant amounts of superoxide. Its distribution is limited in mammals to tissues such as brown adipose and brain, where it is a potentially important site. Two other enzymes involved in fatty acid oxidation, electron transfer flavoprotein and electron transfer flavoprotein quinone oxido reductase may also produce superoxide.

2.5. Endothelial Dysfunction Dysfunction of the endothelium has been implicated in the pathophysiology of different forms of cardiovascular disease, including hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, diabetes, and chronic renal failure [97]. Endothelial dysfunction may be defined as impairment characterized by a shift of the actions of the endothelium toward reduced vasodilation, a proinflammatory state, and prothrombotic setting. Endothelial dysfunction is seen early in the development of atherosclerosis (see chapter 3), before overt vascular and structural changes. It is manifested by impaired vasorelaxation to endothelium-dependent dilators, such as ACh. The pathophysiology of endothelial dysfunction is complex and involves multiple mechanisms. It is characterized by unbalanced concentrations of vasodilating and vasoconstricting factors, the most important being represented by NO and Ang-II, respectively. Nitric oxide is recognized as one of the major mediators of the maintenance of vascular homeostasis, and a decrease in NO bioavailability is associated with endothelial dysfunction. In this context, the causes of reduced vasodilatory responses in endothelial dysfunction include reduced NO generation, oxidative excess and reduced production of hyperpolarizing factor. Reduced NO bioavailability could be due to either reduced formation or accelerated degradation of this

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vasodilator. The mechanism, by which oxidative stress mediates endothelial cell function, and ultimately vascular reactivity, is not fully understood. Although these mechanisms may be multifactorial, there is a growing body of evidence that increased production of ROS may contribute considerably as a causative factor in endothelial dysfunction by reducing NO bioavailability and uncoupling eNOS. The endothelium, the media and also the adventitia produce large amounts of ROS, which will attenuate endothelial mediated dilation, although the mechanisms underlying endothelial dysfunction are located in addition to the endothelium in the smooth muscle cell layer [98]. Superoxide combines with NO, which is synthesized by eNOS, to form peroxynitrite. The consequence is an overall increase in ROS and reduced ability of endothelium-dependent vasodilation. In addition to loss of vasodilation, endothelial dysfunction is associated with endothelial cell apoptosis, increased binding of leukocytes and monocytes, enhanced accumulation of lipid and a predisposition to thrombosis. These events lead to a state of vascular inflammation. Under settings associated with oxidative stress the vasculature per se produces large amounts of superoxide via elevated expression of NADPH oxidase [99]. Consequently, a reduction of NO bioavailability occurs by degrading NO and by forming the highly toxic product peroxynitrite. In addition, ROS formed by activated mononuclear cells can lead to increased expression of cell surface adhesion molecules on endothelium that are considered to be markers of inflammation and thus can enhance the localization and accumulation of additional mononuclear cells, resulting endothelial dysfunction. Formation of ROS by mononuclear cells and the vessel wall may be a link between inflammation and atherosclerosis in hypertensive patients.

2.6. Oxidative Stress and Endothelial Dysfunction in Hypertension A great body of evidence supports the idea that ROS are involved in the pathogenesis of hypertension. Oxidative stress, characterized by increased bioavailability of ROS, plays an important role in the development and progression of cardiovascular dysfunction associated with hypertensive disease. There are many sources of ROS, including neutrophil-like membrane-associated NADPH oxidase, xanthine oxidase, myeloperoxidase, uncoupled eNOS and spillover from mitochondrial respiratory chain [100]. In addition, the occurrence of this disturbance may be caused by decreased antioxidant enzyme activity (SOD, catalase) and reduced levels of ROS scavengers (e.g. vitamin E, glutathione), acting as contributing factors to the development of oxidative stress. These findings are based, in general, on increased levels of plasma thiobarbituric acid-reactive substances and 8-isoprostanes, biomarkers of lipid peroxidation and oxidative stress [68, 101]. Indeed, ROS of vascular origin contribute importantly to peripheral vascular resistance and arterial pressure under pathophysiological conditions such as hypertension [3]. In addition, polymorphonuclear leukocytes and platelets, rich superoxide sources, also participate in vascular oxidative stress and inflammation in hypertensive patients [102, 103]. In this setting, the elevation of blood pressure has been associated with ROS abundance and frequently also with an impairment of endogenous antioxidant mechanisms. Accordingly, increased markers of oxidative stress are found in human hypertensive subjects, as well as in various animal models of hypertension [68, 104107]. Mouse models with genetic deficient in ROS-generating enzymes have lower blood

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pressure compared with wild-type counterparts, and Ang-II infusion fails to induce hypertension in these mice [12, 80]. In addition, in cultured VSMC and isolated arteries from hypertensive rats and humans, ROS production is enhanced, redox-dependent signaling is amplified, and antioxidant bioactivity is reduced [108]. It should be mentioned that in patients with never-treated mild-to-moderate hypertension, lipid peroxidation and oxidative stress were not found increased [109], suggesting that ROS may not be critical in the early stages of human hypertension, but could be more important in severe hypertension. In addition, classical antihypertensive agents such as β-adrenergic blockers, ACE inhibitors, AT1 receptor antagonists, and Ca2+ channel blockers may be mediated, in part, by decreasing vascular oxidative stress [110, 111]. It is of interest to note that increased ROS production in vascular tissues has also effects other than elevation of blood pressure. Particularly superoxide anions, has been implicated as playing an important role in vascular events such as vascular remodeling after angioplasty, atherosclerosis, myocardial infarction, and ischemic stroke [65]. Thus, therapeutic strategies should aim to restore the bioavailability of NO, either scavenging ROS or through down-regulation of their generation and/or up-regulation of eNOS activity and antioxidant enzymes.

3. Antioxidants in Hypertension With the recent advances in our understanding of the complexity of oxidative stress and redox signaling in the vascular system pointing to a central role of oxidative stress in the pathogenesis of vascular dysfunction, it has arisen a growing interest regarding the therapeutic possibilities to target ROS in the management of hypertension and other cardiovascular diseases. The deleterious effects resulting from the formation of ROS are, to a large extent, prevented by various antioxidant systems. Theoretically, agents that reduce oxidant formation should be more efficacious than nonspecific inefficient antioxidant scavengers in ameliorating oxidative stress. Therefore, it should be expected a beneficial effect derived from several antioxidants, such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione, BH4, and N-acetylcysteine, among others, which have shown to improve endothelial function and NO bioaction in cultured cells, and in animal and human clinical studies of vascular reactivity. In support of this view, epidemiological studies suggest that individuals with higher antioxidant intake have reduced cardiovascular risk. Based on experimental evidence of the importance of oxidative stress in vascular damage, there has been great interest in developing strategies that target ROS in the treatment of hypertension and other cardiovascular diseases. Therapeutic approaches that have been considered include mechanisms to increase antioxidant bioavailability or to reduce ROS generation by decreasing activity of superoxide-generating enzymes. Gene therapy targeting oxidant systems are also being developed, but their use in clinical hypertension remains unclear. This section presents the available evidence for the potential of antioxidants in the prevention and treatment of hypertension associated with oxidative stress, as supported by experimental investigations, observational findings, clinical trials, and epidemiological data with special reference to the antihypertensive effect of the main antioxidants of human use.

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3.1. Vitamin C Vitamin C (ascorbic acid) is a potent water-soluble antioxidant in humans. It is a sixcarbon lactone synthesized from glucose in the most mammalian species, mainly in liver, but not in humans. Vitamin C is an electron donor and therefore a reducing agent. When ascorbate acts as an antioxidant or enzyme cofactor, it becomes oxidized to dehydroascorbic acid. The latter can be used by cells to regenerate ascorbate, and directly or indirectly, it can change the redox state of many other molecules. Vitamin C performs against oxidation of lipids, proteins and DNA, subsequently protecting their structure and biological function. In addition, on the vascular wall vitamin C behaves as enzyme modulator exerting up-regulation on eNOS and down-regulation of NADPH oxidase [112]. Recently, it was demonstrated that vitamin C inhibits the effects of ET-1 of impairing endothelium-dependent and endotheliumindependent vasodilation and the stimulation of interleukin-6 (IL-6) release in humans in vivo. This suggests that the mechanism by which ET-1 impairs vascular function and stimulates release of IL-6 involves increased oxidative stress [113]. Most studies have demonstrated an inverse relationship between plasma ascorbate levels and blood pressure in both normotensive and hypertensive populations [68, 114]. In a recent study, a decreasing trend was observed with vitamin C levels and risk of hypertension in women but not in men [115]. Vitamin C supplementation is associated with reduced blood pressure in hypertensive patients, with systolic blood pressure falling by 3.6–17.8 mmHg for each 50 µmol/l increase in plasma ascorbate [68, 116, 117]. Nevertheless, there are several small and short-term clinical trials in which the effect of vitamin C supplements on blood pressure have yielded inconsistent findings [116, 118-120]. The lack of antihypertensive efficacy observed in studies using supplementation with vitamin C alone could be due to the pharmacokinetics of vitamin C and/or the decreased bioavailability of NO under conditions of oxidative stress. The antihypertensive effect of vitamin C is expected to occur at 10 mmol/L, a plasma concentration unobtainable in humans following oral administration. However, this concentration is required to compete efficiently with the reaction of NO with superoxide, due to their high reaction rate constant, which is even higher than the reaction between SOD and NO [121]. The lack of a therapeutic antihypertensive plasma vitamin C concentration via oral administration may be due to its renal threshold at doses between 60 and 100 mg/day. The steady-state concentration of vitamin C is attained at approximately 80 µmol/L, and plasma is completely saturated at daily doses of over 400 mg [122]. Pharmacokinetic modeling indicates that, with oral administration, even at very large and frequent doses of vitamin C, plasma concentrations will only be increased modestly, from 70 µmol/L to a maximum of 220 µmol/L, whereas intravenous administration increases it as high as 14 mmol/L [123]. Thus the antihypertensive effect may only occur in plasma following infusion of high vitamin C doses. Accordingly, intra-arterial administration of vitamin C has been shown to cause a decrease in blood pressure in subjects with essential hypertension [124]. The molecular mechanisms underlying the in vivo antioxidant effects of vitamin C related with blood pressure modulation are not fully understood. Nevertheless, it was shown that these effects are mediated in part by the ability of vitamin C to protect BH4 from oxidation and thereby increase the enzymatic activity of eNOS. It should be noted that BH4 is a cofactor necessary

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for NO generation via eNOS, otherwise becoming uncoupled, a form now recognized as an important source of superoxide rather than NO [125], a condition likely to occur under a prooxidant state. A hypothesis for the mechanisms whereby vitamin C, as well as other antioxidants, could exert antihypertensive effects is shown in Figure 2-1.

Figure 2-1. Involvement of vitamin C, as well as other antioxidants, in counteracting the elevation of blood pressure induced by oxidative stress. BH4, tetrahydrobiopterin; oxBH4, oxidized tetrahydrobiopterin; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; Ang-II, angiotensin II; AT1, angiotensin II type 1 receptors; AT1-AA, autoantibodies to angiotensin II type 1 receptors; U-II, human urotensin II; UT, human urotensin II receptors; ET-1, endothelin-1; ETA, type A endothelin-1 receptors; PGI2, prostacyclin; EDCF, endothelium derived contracting factor; TP, thromboxaneprostaglandin receptors. The effects of vitamin C in these pathways are indicated as down-regulation ) and up-regulation ( ( 127)[143].

). (Adapted from Rodrigo et al., Fund Clin Pharmacol 2007a; 21: 111-

3.2. Vitamin E Vitamin E is a major lipid-soluble antioxidant that has received considerable attention. Epidemiological data support a role of high dietary vitamin E intake and a reduced incidence of cardiovascular disease [126]. Tocopherols have been shown to increase PGI2 levels in

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endothelial cells via opposing effects on phospholipase A2 and cyclo-oxygenase 2 [127], a potential beneficial effect against endothelial dysfunction as PGI2 is a prostanoid vasodilator which is important for maintaining normal vascular function. Increasing evidence indicates that vitamin E can act as a biological modifier independently of its antioxidant activity. Experimental evidence available shows that vitamin E is capable of dose-dependently regulating mitochondrial generation of superoxide and hydrogen peroxide. This effect is reached through the prevention of electron leakage, by mediating the superoxide generation systems directly and/or by scavenging superoxide generated. By down-regulating mitochondrial generation of superoxide and related ROS, vitamin E not only attenuates oxidative damage but also modulates the expression and activation of signal transduction pathways and other redox-sensitive biological modifiers [128]. However, intervention trials have not been convincing, with a number of studies demonstrating no beneficial effect of vitamin E on cardiovascular disease outcomes [129-132]. Most of these studies have generally not reported blood pressure outcomes, although a subset of the Primary Prevention Project (PPP) study [133] did show no effect of vitamin E supplementation on clinic or ambulatory blood pressure in treated hypertensive patients. Moreover, a meta-analysis has highlighted an increase in all-cause mortality with high-dose vitamin E supplementation [134]. In support of this view, other study using supplementation with vitamin E, either as atocopherol or mixed tocopherols, showed a significant increase in blood pressure, pulse pressure and heart rate in individuals with type 2 diabetes. These increases were observed despite a reduction in plasma total F2-isoprostanes [135]. It should be noted that although vitamin E can inhibit LDL oxidation in vitro, it is unlikely to achieve sufficiently high concentrations in the vascular microenvironment to interfere effectively with all components of oxidative stress, and has limited activity against superoxide and peroxynitrite driven processes [136]. Therefore, taken together these data support the view that vitamin E alone supplements at daily doses over 400 IU may increase all-cause mortality and should be avoided. It is of interest to note that the association of vitamins C and E is expected to have an antihypertensive effect probably due to the fact that this combined therapy provides a reinforcement of their individual properties through a complementary effect in improving endothelial dysfunction [137]. Both vitamins C and E not only behave as scavengers of ROS, but also are able to induce the down-regulation of NADPH oxidase and the up-regulation of eNOS [112, 138]. Vitamin C may reduce the α-tocopheroxyl radical, thereby abrogating lipid peroxidation [139] and further supporting an antihypertensive effect for this association. The use of these association aimed to cause an antihypertensive effect is discussed below.

3.3. Clinical Trials for Association of Vitamins C and E Despite the biological effects of both vitamin C and E, as shown by experimental models, long-term clinical trials have failed to consistently support their antihypertensive effects in patients at high cardiovascular risk. Most of clinical studies have looked at all-cause or cardiovascular mortality, rarely focusing on blood pressure as a primary end point [140], but none of the large clinical trials examined the effects of antioxidants specifically on blood

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pressure [107]. Some short-term trials have shown that supplemental antioxidant vitamin intake lowers blood pressure [118, 141, 142] but the majority of clinical trials did not find any antihypertensive effects of antioxidant vitamins. However, most of these studies lack rigorous exclusion criteria in the selection of subjects to avoid the influence of confounders [143]. It deserves special mention that regarding cohorts included in large trials, most subjects had irreversible cardiovascular disease. Some of these alterations could contribute to perpetuate the increased ROS production by the vascular wall. Thus, in atherosclerotic arteries there is evidence for increased expression of the NADPH oxidase subunit gp91phox and Nox4, all of which may contribute to increased oxidative stress within vascular tissue [144]. In addition, in this setting there is an increase in the expression of the Ang-II receptor subtype AT1, providing evidence for stimulation of the renin angiotensin system and simultaneously for an activation of the NADPH oxidase in the arterial wall [145]. Recently, a randomized double-blind placebo-controlled study was conducted to test the hypothesis that oral administration of vitamins C and E together, by improving the antioxidant status, causes a decrease in blood pressure in patients with mild-to-moderate essential hypertension [146]. The results of this study, performed with newly diagnosed hypertensives, without end-organ damage, showed for the first time a specific association between oxidative-stress-related parameters and blood pressure, thus suggesting a role of oxidative stress in the pathogenesis of essential hypertension. Moreover, the concomitant decrease in blood pressure and oxidative stress raises the possibility that oral administration of vitamins C + E in patients with essential hypertension may be considered as an adjunct therapy for hypertension in those patients. In summary, the available data lead us to think in a beneficial antihypertensive effect of vitamins C and E if administered during the phase of endothelial dysfunction, which precedes an established vascular damage. In this setting it would be more likely to successfully reverse, or at least counteract, the deleterious effects of ROS on the vascular wall. In contrast, it should not be expected an antihypertensive effect in patients having significant cardiovascular disease, in which case chronic damaging effects of oxidative stress may be irreversible.

3.4. N-Acetylcysteine The antioxidant N-acetyl-L-cysteine (NAC), a sulfhydryl group donor, improves renal dysfunction and markedly decreases arterial pressure and renal injury in Dahl salt-sensitive hypertension [147]. In an experimental model of hypertension, systolic blood pressure was significantly higher in rats with 10% glucose feeding for 20 weeks [148]. This was associated with a higher production of superoxide anion and NADPH oxidase activity in aorta. The therapeutic effects of NAC in rats with established L-NAME hypertension were less pronounced than the preventive effects of NAC on the development of L-NAME hypertension [149]. Similarly, in spontaneously hypertensive rats, chronic administration of NAC partially attenuated the blood pressure increase in young rats, while its effect was negligible in adults with fully developed hypertension. These results suggest that the inhibition of the oxidative stress in hypertensive states contributes to the therapeutic effects of NAC; it seems that ROS play a more important role in the induction than in the

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maintenance of hypertension. On the other hand, in patients with type 2 diabetes and hypertension, oral supplementation of NAC + L-arginine for 6 months caused a reduction of both systolic and diastolic mean arterial blood pressure [150]. NAC administered intravenously during hemodialysis reduced ADMA levels more significantly than hemodialysis alone [151]. In relation to the mechanisms accounting for these results, the effect of NAC may be mediated by an NO-dependent mechanism, probably through the protective effect of NAC on NO oxidation. In patients with type 2 diabetes NAC improves NO bioavailability via reduction of oxidative stress and increase of NO production. NAC augments the levels of reduced glutathione and enhances the activity of NOS, probably by protecting its essential cofactor BH4 from oxidation by the excess superoxide. Moreover, NAC has been shown to protect the sulfhydryl groups of NOS from destruction by free radicals and thus to maintain its activity [152]. These data are consistent with the NACinduced enhancement of the hypotensive effect of angiotensin-converting enzyme inhibitors, as it is an effect at least partially mediated by NO. Therefore, NAC could be considered as an adjuvant in the pharmacology of antihypertensive drugs having antioxidant properties and/or acting through an improvement of NO bioactivity.

3.5. Polyphenols Polyphenols are the most abundant antioxidant in the diet. Their intake is 10 times higher than vitamin C and 100 times higher than vitamin E or carotenoids. Polyphenols like catechin or quercetin can directly scavenge ROS, such as superoxide, hydrogen peroxide, or hypochlorus acid, which can be very deleterious by damaging lipids, proteins and DNA. The phenolic core can act as a buffer and capture electrons from ROS to render them less reactive. On one hand, flavonoids are the major constituents of this group with more than 4000 compounds. On the other hand, the non-flavonoids compounds contain an aromatic ring with one or more hydroxyl group. This group includes stilben (resveratrol), phenolic acids (gallic acid) saponin (ginsenoside) and other polyphenols like curcumin and tannins. The role of polyphenols in plants may partly explain the biological properties observed in vitro or in vivo: they are involved in defense against infection and confer protective effects to the plants against stress, such as ultraviolet radiation, pathogens and physical damages [153]. Epidemiological studies have shown an inverse correlation between polyphenols enriched diet and reduced risks of cardiovascular diseases [154]. In humans, 30 min after the consumption of red wine or polyphenols (1 g/kg body weight), circulating NO concentration increases to 30 and 40 nM, respectively. Chronic treatment with red wine polyphenols reduces hypertension and vascular dysfunction through reduction in vascular oxidative stress in female spontaneously hypertensive rats in a manner independent of the ovarian function [155]. A reduction of the blood pressure (11 mmHg) and an increase of heart rate are observed [156]. In hypertensive patients, the use of olive oil can reduce the blood pressure [157]. Short-term oral administration of red wine polyphenols produces a decrease in blood pressure in normotensive rats. This haemodynamic effect was associated with an enhanced endothelium-dependent relaxation and an induction of gene expression within the arterial wall, which together maintain unchanged agonist-induced contractility [158]. In addition, red

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wine polyphenols can accelerate the regression of blood pressure and improves structural and functional cardiovascular changes produced by chronic inhibition of NO synthesis [159]. The flavonol quercetin, one of the most abundant polyphenolic compounds found in the human diet, relaxes vascular smooth muscle and its chronic daily treatment reduced blood pressure and endothelial dysfunction in experimental models of hypertension characterized by an activation of the renin–angiotensin system, such as in spontaneously hypertensive rats [160, 161], in rats made hypertensive by chronic inhibition of NO synthase [162] or in renovascular hypertensive rats [163]. In relation to the mechanism whereby polyphenols reduce the blood pressure, it has been reported an effect on the endothelium mainly due to NO production [164, 165]. Thus, the beneficial effects of plant polyphenols in prevention of hypertension may result from their complex influence on the NO balance in the cardiovascular system. The mechanism of endothelial NO release elicited by polyphenols has been investigated. Red wine polyphenols can modulate the production of NO through an extracellular Ca2+dependent mechanism in endothelial cells. Resveratrol and quercetin have been shown to induce an increase of the intracellular concentration of Ca2+, by activation K+ channels or inhibition of Ca2+-ATPases of the endoplasmic reticulum in endothelial cells. Prevention of both blood pressure increase and cardiovascular remodeling by chronic treatment with the antioxidant provinol was associated with increased NO synthase activity and enhanced expression of endothelial NO synthase [46]. It has also been documented that polyphenols of red wines strongly inhibit the synthesis of ET-1, a vasoactive peptide that is crucial for the development of coronary atherosclerosis [166]. These data suggest that reduced oxidative stress due to antioxidant action of provinol, its ability to increase endothelial NO synthase activity and to decrease ET-1 synthesis may contribute to the polyphenol-induced antihypertensive effect and protection against cardiovascular remodeling in NO-deficient rats [167]. Nevertheless, the antioxidant treatment is expected to be more efficient in the prevention than in the reduction of established hypertension [168].

3.6. Diet There is sufficient evidence to suggest that dietary approaches may help to prevent and control high blood pressure. There are dietary approaches regarding the prevention and management of hypertension: i.e. moderate use of sodium, alcohol, an increased potassium intake, plant fibers, calcium (and dairy products) and adherence to healthy dietary patterns such as Dietary Approaches to Stop Hypertension (DASH) [169]. In addition, the study also presents evidence regarding other nutritional factors which may possibly be associated with levels of blood pressure, but for which there is as yet insufficient current scientific evidence to support the issue of specific dietary recommendations. The Mediterranean diet has been described by the following characteristics: an abundance of plant foods (fruits, vegetables, breads, other forms of cereals, potatoes, beans, nuts, and seeds); minimally processed, seasonally fresh, and locally grown foods; fresh fruit as the typical daily dessert, with sweets containing concentrated sugars or honey consumed only a few times per week; olive oil as the main source of fat; dairy products (principally cheese and yogurt) only in low-tomoderate amounts; red meat in low amounts; and wine, usually red wine, in low-to moderate

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amounts, normally with meals. In a Mediterranean population with an elevated fat consumption, a high fruit and vegetable intake is inversely associated with blood pressure levels [170]. In the clinical trial DASH, assessing the effects of dietary patterns on blood pressure [169], it was demonstrated that certain dietary patterns can favorably affect blood pressure in adults with average systolic blood pressures of less than 160 mmHg and diastolic blood pressures of 80 to 95 mmHg. Specifically, a diet rich in fruits, vegetables, and low-fat dairy products and with reduced saturated and total fat lowered systolic blood pressure by 5.5 mmHg and diastolic blood pressure by 3.0 mmHg more than a control diet. More recently, it was suggested that adhering to a Mediterranean-type diet could contribute to the prevention of age-related changes in blood pressure [171].

4. Conclusions and Perspectives There is considerable evidence supporting the view that oxidative stress is involved in the pathophysiology of hypertension. Indeed, ROS are mediators of the major physiological vasoconstrictors, such as Ang-II, ET-1, and U-II. In turn, oxidative stress, characterized by increased bioavailability of ROS, plays an important role in the development and progression of cardiovascular dysfunction associated with hypertensive disease. There are many sources of ROS, including the enzymatic components of the vascular wall itself. In addition, the effects of classical antihypertensive agents such as β-adrenergic blockers, ACE inhibitors, AT1 receptor antagonists, and Ca2+ channel blockers may be mediated, in part, by decreasing vascular oxidative stress. However, despite the consistent and promising findings from experimental investigations, the clinical trials, and epidemiological data suggesting the use of antioxidants as antihypertensive agents, data are inconclusive. It appears difficult to reconcile these negative studies in view of the large body of evidence supporting the role of oxidative stress in cardiovascular disease. First, despite scavenging free radicals is indeed the best way to decrease ROS bioavailability, in the particular case of the cardiovascular system the efficacy of this intervention is limited by the high rate reaction constant between superoxide anion and NO. Second, most of the clinical trials were performed with heterogeneity of studied populations, inappropriate or insensitive methodologies to evaluate oxidative state, and incorrect antioxidant therapies. Third, patients with significant cardiovascular disease were enrolled; therefore, these patients were with established vascular damage in such way that it could contribute to perpetuate the increased ROS production by the vascular wall. The use of either vitamin C or vitamin E alone has not proved antihypertensive therapeutic efficacy; but their association, as well as with other antioxidants has been more promising, likely due to a synergistic effect. The most relevant effect has been shown by the use of diets rich in antioxidants provided by fruits and vegetables and low fat. Although the role of antioxidant therapy for primary prevention remains an open question, it could be concluded that all these interventions would be expected to be more efficient in the prevention than in the reduction of established hypertension.

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[134] Miller ER III, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Metaanalysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann. Intern. Med. 2005;142:37–46. [135] Ward NC, Wu JH, Clarke MW, Puddey IB, Burke V, Croft KD, Hodgson JM. The effect of vitamin E on blood pressure in individuals with type 2 diabetes: a randomized, double-blind, placebo-controlled trial. J. Hypertens. 2007;25:227-234. [136] Münzel T, Keaney JF Jr. Are ACE inhibitors a “magic bullet” against oxidative stress? Circulation. 2001;104:1571–1574. [137] Bilodeau JF, Hubel CA. Current concepts in the use of antioxidants for the treatment of preeclampsia. J. Obstet. Gynaecol. Can. 2003;25:742–750. [138] Attia DM, Verhagen AM, Stroes ES, van Faassen EE, Gröne HJ, De Kimpe SJ, Koomans HA, Braam B, Joles JA. Vitamin E alleviates renal injury, but not hypertension, during chronic nitric oxide synthase inhibition in rats. J. Am. Soc. Nephrol. 2001;12:2585–2593. [139] Heller R, Werner-Felmayer G, Werner ER. Antioxidants and endothelial nitric oxide synthesis. Eur. J. Clin. Pharmacol. 2006;62(Suppl. 13):21–28. [140] Ward NC, Croft KD. Hypertension and oxidative stress. Clin. Exp. Pharmacol. Physiol. 2006;33:872-876. [141] Mullan B, Young IS, Fee H, McCance DR. Ascorbic acid reduces blood pressure and arterial stiffness in type 2 diabetes. Hypertension. 2002;40:804–809. [142] Galley HF, Thornton J, Howdle PD, Walker BE, Webster NR. Combination oral antioxidant supplementation reduces blood pressure. Clin. Sci. 1997;92:361–365. [143] Rodrigo R, Guichard C, Charles R. Clinical pharmacology and therapeutic use of antioxidant vitamins. Fundam. Clin. Pharmacol. 2007a;21:111-127. [144] Sorescu D, Weiss D, Lassègue B, Clempus RE, Szöcs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK. Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation. 2002;105:1429–1435. [145] Nickenig G, Bäumer AT, Temur Y, Kebben D, Jockenhövel F, Böhm M. Statinsensitive dysregulated AT1 receptor function and density in hypercholesterolemic men. Circulation. 1999;100:2131–2134. [146] Rodrigo R, Prat H, Passalacqua W, Araya J, Bächler JP. Decrease in oxidative stress through supplementation of vitamins C and E is associated with a reduction in blood pressure in patients with essential hypertension. Clin. Sci. (Lond) 2008;114:625-634. [147] Tian N, Rose RA, Jordan S, Dwyer TM, Hughson MD, Manning RD Jr. NAcetylcysteine improves renal dysfunction, ameliorates kidney damage and decreases blood pressure in salt-sensitive hypertension. J. Hypertens. 2006;24:2263-2270. [148] El Midaoui A, Ismael MA, Lu H, Fantus IG, de Champlain J, Couture R. Comparative effects of N-acetylcysteine and ramipril on arterial hypertension, insulin resistance, and oxidative stress in chronically glucose-fed rats. Can. J. Physiol. Pharmacol. 2008;86:752-760. [149] Rauchová H, Pechánová O, Kunes J, Vokurková M, Dobesová Z, Zicha J. Chronic Nacetylcysteine administration prevents development of hypertension in N(omega)-nitro-

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L-arginine methyl ester-treated rats: the role of reactive oxygen species. Hypertens. Res. 2005;28:475-482. [150] Martina V, Masha A, Gigliardi VR, Brocato L, Manzato E, Berchio A, Massarenti P, Settanni F, Della Casa L, Bergamini S, Iannone A. Long-term N-acetylcysteine and Larginine administration reduces endothelial activation and systolic blood pressure in hypertensive patients with type 2 diabetes. Diabetes Care. 2008;31:940-944. [151] Thaha M, Widodo, Pranawa W, Yogiantoro M, Tomino Y. Intravenous Nacetylcysteine during hemodialysis reduces asymmetric dimethylarginine level in endstage renal disease patients. Clin. Nephrol. 2008;69:24-32. [152] Zembowicz A, Hatchett RJ, Radziszewski W, Gryglewski RJ. Inhibition of endothelial nitric oxide synthase by ebselen. Prevention by thiols suggests the inactivation by ebselen of a critical thiol essential for the catalytic activity of nitric oxide synthase. J. Pharmacol. Exp. Ther. 1993;267:1112-1118. [153] Middleton E Jr, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 2000;52;673–751. [154] Ulbricht TL, Southgate DA: Coronary heart disease: seven dietary factors. Lancet. 1991;338:985–992. [155] López-Sepúlveda R, Jiménez R, Romero M, Zarzuelo MJ, Sánchez M, Gómez-Guzmán M, Vargas F, O'Valle F, Zarzuelo A, Pérez-Vizcaíno F, Duarte J. Wine polyphenols improve endothelial function in large vessels of female spontaneously hypertensive rats. Hypertension. 2008;51:1088-1095. [156] Matsuo S, Nakamura Y, Takahashi M, Ouchi Y, Hosoda K, Nozawa M, Kinoshita M. Effect of red wine and ethanol on production of nitric oxide in healthy subjects. Am. J. Cardiol. 2001;87:1029-1031. [157] Ferrara LA, Raimondi AS, d’Episcopo L, Guida L, Dello Russo A, Marotta T: Olive oil and reduced need for antihypertensive medications. Arch. Intern. Med. 2000;160:837– 842. [158] Diebolt M, Bucher B, Andriantsitohaina R: Wine polyphenols decrease blood pressure, improve NO vasodilatation, and induce gene expression. Hypertension. 2001;38:159– 165. [159] Bernátová I, Pechánová O, Babál P, Kyselá S, Stvrtina S, Andriantsitohaina R. Wine polyphenols improve cardiovascular remodeling and vascular function in NO-deficient hypertension. Am. J. Physiol. Heart Circ. Physiol. 2002;282:H942–948. [160] Machha A, & Mustafa MR. Chronic treatment with flavonoids prevents endothelial dysfunction in spontaneously hypertensive rat aorta. J. Cardiovasc. Pharmacol. 2005;46:36–40. [161] Sánchez M, Galisteo M, Vera R, Villar IC, Zarzuelo A, Tamargo J, Pérez-Vizcaíno F, Duarte J. Quercetin downregulatesNADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats. J. Hypertens. 2006;24:75–84. [162] Duarte J, Jiménez R, O'Valle F, Galisteo M, Pérez-Palencia R, Vargas F, PérezVizcaíno F, Zarzuelo A, Tamargo J. Protective effects of the flavonoid quercetin in chronic nitric oxide deficient rats. J. Hypertens. 2002;20:1843–54.

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[163] García-Saura MF, Galisteo M, Villar IC, Bermejo A, Zarzuelo A, Vargas F, Duarte J. Effects of chronic quercetin treatment in experimental renovascular hypertension. Mol. Cell Biochem. 2005;270(1-2):147-55. [164] Duarte J, Andriambeloson E, Diebolt M, Andriantsitohaina R: Wine polyphenols stimulate superoxide anion production to promote calcium signaling and endothelialdependent vasodilatation. Physiol. Res. 2004;53:595–602. [165] Zenebe W, Pechanova O, Andriantsitohaina R: Red wine polyphenols induce vasorelaxation by increased nitric oxide bioactivity. Physiol. Res. 2003;52:425–432. [166] Corder R, Douthwaite JA, Lees DM, Khan NQ, Viseu Dos Santos AC, Wood EG, Carrier MJ. Endothelin-1 synthesis reduced by red wine. Nature. 2001;414(6866):863864. [167] Pechánová O, Rezzani R, Babál P, Bernátová I, Andriantsitohaina R. Beneficial effects of Provinols: cardiovascular system and kidney. Physiol. Res. 2006;55 Suppl 1:S17-30. [168] Pechánová O, Zicha J, Paulis L, Zenebe W, Dobesová Z, Kojsová S, Jendeková L, Sládková M, Dovinová I, Simko F, Kunes J. The effect of N-acetylcysteine and melatonin in adult spontaneously hypertensive rats with established hypertension. Eur. J. Pharmacol. 2007;561(1-3):129-136. [169] Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM, Bray GA, Vogt TM, Cutler JA, Windhauser MM, Lin PH, Karanja N. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N. Engl. J. Med. 1997;336:1117-1124. [170] Alonso A, de la Fuente C, Martín-Arnau AM, de Irala J, Martínez JA, MartínezGonzález MA. Fruit and vegetable consumption is inversely associated with blood pressure in a Mediterranean population with a high vegetable-fat intake: the Seguimiento Universidad de Navarra (SUN) Study. Br. J. Nutr. 2004;92:311-319. [171] Núñez-Córdoba JM, Valencia-Serrano F, Toledo E, Alonso A, Martínez-González MA. The Mediterranean Diet and Incidence of Hypertension: The Seguimiento Universidad de Navarra (SUN) Study. Am. J. Epidemiol. 2008 Nov 26.

In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter III

Atherosclerosis Víctor Molina1 and Ramón Rodrigo2 1

Faculty of Medicine, University of Chile Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile Supported by FONDECYT, grant 1070948 2

Abstract Atherosclerosis is a major source of mortality, being the underlying cause for most cases of cardiovascular diseases such as ischemic heart disease and cerebrovascular disease. Reactive oxygen species (ROS) can regulate several cellular processes, having a key role in the homeostasis of the vascular wall. There is compelling evidence pointing to ROS as important factors for the development of atherosclerosis. Many of the proatherogenic actions of ROS occur through the generation of oxidized LDL. Also, ROS can contribute to the development of endothelial dysfunction through the consumption of nitric oxide and generation of peroxynitrite. Endothelial dysfunction constitutes an early feature of atherogenesis, preceding the alterations that later perpetuate the lesion formation. Atherogenesis includes several processes, such as accumulation and oxidation of LDL in the subendothelial space, expression of adhesion molecules and chemoattractant mediators, adhesion of monocytes, generation of foam cells, production of inflammatory mediators and proliferation of certain cell types. Since most of these processes can be modulated by ROS, supplementation with antioxidants is expected to exert some degree of protection against atherosclerosis. Several lines of evidence support a role of antioxidant supplementation in attenuating some of the processes involved in atherogenesis. However, clinical trials have failed to consistently prove a protective effect. The potential role of antioxidant supplementation against atherosclerosis development or progression remains an open question.

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1. Introduction Atherosclerosis is a major source of mortality, being the underlying condition for most cases of ischemic heart disease and cerebrovascular disease that constitute the leading causes of death worldwide [1, 2]. Only in USA, these two conditions caused 589,266 deaths in 2005 [3]. Cardiovascular diseases (CVD) are expected to be the first cause of death in almost every country in the upcoming years [2, 4]. The process of atherosclerosis is characterized by the accumulation of macrophages within the wall of large and medium sized arteries, and proliferation of certain cell types. The consequence is the formation of a lesion known as atheromatous plaque that starts early in life, even in late childhood [5], and progressively occludes the vessel lumen. Among the complications that this plaque can suffer it is a rupture leading to thrombosis and acute impaired blood supply in certain organs such as heart and brain, resulting in heart attack and stroke, respectively. Reactive oxygen species (ROS) have a key role in regulating several cellular processes as well as homeostasis of the vascular wall. As shown in this book, oxidative stress has been implicated in the pathogenesis of multiple highly prevalent diseases. Accordingly, there is compelling evidence pointing to ROS as important factors in the development of atherosclerosis. Understanding the pathophysiological basis of ROS involvement in atherogenesis is mandatory for the design of future therapeutic approaches aimed to prevent or treat this disease. This chapter focuses on the available data that supports a role of oxidative stress in the mechanism of production and perpetuation of atherosclerosis, as well as the current evidence regarding the use of antioxidants in the prevention or treatment of this pathology.

2. Pathophysiology of Atherosclerosis The pathophysiology of atherosclerosis constitutes an extensive subject closely related to the current available evidence of ROS involvement in atherogenesis. This section starts by referring to the known risk factors for atherosclerosis, followed by the morphologic features of the atherosclerotic lesion and the hypotheses that have been postulated to explain its initiation. Finally, the role of the different components involved in the atherogenesis process and their relation to ROS is reviewed in more detail.

2.1. Risk Factors Several variables have been associated with an increased chance of developing CVD. Most of these factors have arisen from the analysis of poblational studies relating the incidence of CVD to the presence of other conditions. One of the major sources of information on this topic is the Framingham Heart Study, a cohort follow up that started in 1948. Recognizing these risk factors has made possible the selection of those with a stronger association and the development of cardiovascular risk prediction charts that facilitate the

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patient assessment in the clinical practice [6, 7, 8]. Also, it has led to the design of guidelines for the prevention of CVD, focusing on these factors [8, 9]. Age Age is one of the strongest risk factors for CVD, and, although it is a non modifiable one, it has a special relevance due to its unavoidability. Based on the Framingham Heart Study data, the average risk of developing coronary heart disease during the following 10 years is 3% for a 30-34 years old male, increasing to 14% for a 50-54 years old and to 21% for a 6064 years old [6]. Gender Males exhibit a higher risk in relation to same age females [6]. Serum Cholesterol High levels of total cholesterol and LDL cholesterol have been consistently associated with higher cardiovascular risk [6]. The treatment with cholesterol lowering drugs diminishes the risk in these patients, especially with drugs such as statins, highly effective in lowering levels of LDL cholesterol [10]. On the contrary, high levels of HDL cholesterol are cardioprotective, whereas low levels associate with an increased cardiovascular risk [11, 12]. Blood Pressure Increased blood pressure is considered a major risk factor for CVD [13]. Pharmacological reduction of blood pressure is consistently associated with a reduction in total cardiovascular mortality [14]. Diabetes Mellitus Macrovascular complication of diabetes, including diseases of coronary arteries, carotid arteries and peripheral vessels, are an important cause of mortality by CVD in diabetic patients [15]. There are several lines of evidence supporting a role of diabetes mellitus in the pathogenesis of atherosclerosis [15]. Obesity Obesity, and particularly abdominal obesity, increases cardiovascular risk, either by predisposing to other risk factors, such as diabetes mellitus or hypertension, or as an independent predictor of CVD [16]. Smoking Cigarette smoking is an important risk for CVD. Smoking cessation results in a reduction of this risk [17].

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2.2. Morphologic Features of Atherosclerosis The normal arterial wall is formed by three concentric layers surrounding the arterial lumen. The layer closest to the lumen is called the intima, the middle layer is known as the media and the most external layer is the adventitia. There are also two concentric layers of elastin that separate these three structures. The internal elastic lamina separates the intima from the media, and the external elastic lamina separates the media from the adventitia. The luminal surface of arteries is formed by a single layer of endothelial cells, delimitated by its basal membrane, which is in contact with the internal elastic lamina. Endothelial cells are the main cell type present in the intima, although vascular smooth muscle cells (VSMC) and macrophages can be found occasionally. The thickness of the intima is not uniform and can be expressed as the intima:media ratio, which is normally between 0,1 to 1 [18]. Besides establishing a structural barrier for the blood flow, the endothelium is implicated in the regulation of many processes such as vascular tone, thrombosis and inflammation, whose relevance in the pathogenesis of atherosclerosis will be discussed later in this chapter. The media is composed mainly by layers of VSMC, the number of which increases with the arterial size. Vascular smooth muscle cells are held together by an extracellular matrix of elastic fibers and collagen. The adventitia is a loose matrix of fibroblasts, VSMC and collagen; its potential role in the pathogenesis of atherosclerosis through the production of ROS will be discussed later. The morphologic manifestation of atherosclerosis is the presence of a lesion known as atheromatous plaque or simply plaque. The developing lesion evolves during many years before it can become symptomatic. Atherosclerotic lesions have been extensively characterized and classified [19, 20] in six types, according to morphologic and histologic features that reflect a progression in severity. These lesions form first in some regions of arteries, such as bifurcations, that show a physiologic increase of the intimal thickness known as adaptative intimal thickening, particularly its eccentric form [18]. Accordingly, these susceptible regions have been called atherosclerosis-prone areas [18]. Atherosclerotic lesions are divided in initial lesions (type I-II), intermediate lesions (type III) and advanced lesions (type IV-VI). Type I lesions are the first microscopically and chemically detectable lipid deposits in the intima, characterized by the presence of small and isolated groups of lipid loaded macrophages (foam cells). Type II lesions include “fatty streaks” as a macroscopic lesion and are characterized by a greater number of foam cells and by the presence of intimal VSMC with lipid inclusions. Type III lesions are intermediate lesions between type II lesions and advanced lesions, being characterized by the presence of isolated pools of extracellular lipid deposits. Type IV lesions are known as atheroma, and are considered advanced lesions. They present a dense accumulation of extracellular lipid called the lipid core, formed by confluence of extracellular isolated lipid deposits present in type III lesions. The lipid core is separated from the arterial lumen by a thin tissue layer. Type V lesions are characterized by the presence of a prominent fibrous connective tissue formation. They present a thick fibrotic cover separating the lipid core from the arterial lumen, known as the fibrous cap. Type VI lesions present a more complex morphology, with surface disruption, hematoma, hemorrhage and/or thrombosis.

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As described in the previously exposed classification, mature plaques have a lipid core that consists in accumulation of extracellular lipid. The lipid core is separated from the arterial lumen by the fibrous cap. The region where the fibrous cap contacts the normal arterial wall is called the “shoulder”. This region of the plaque is more cellular than other areas, presenting a high number of macrophages, VSMC and T-lymphocytes. The shoulder region is where the majority of the plaque ruptures occur. In relation to the possibility of rupture, mature plaques can be classified in stable and unstable. Stable plaques have a thicker fibrous cap, smaller lipid core and shoulder region with a less inflammatory component than unstable plaques. In consequence, unstable plaques are weaker structurally and more prone to rupture.

2.3. Hypotheses for the Development of Atherosclerosis Several hypotheses have been proposed to explain the pathogenesis of atherosclerosis. During the nineteenth century Rudolf Virchow proposed that atherosclerotic lesions were the result of an injury to the arterial wall, involving inflammatory and proliferative responses that preceded the development of advanced lesions [21]. A modification of Virchow hypothesis by Ross and Glomset led to the establishment of the original response-to-injury hypothesis [21, 22]. These authors proposed that an initial step in the developing of atherosclerotic lesions would be an injury that resulted in the desquamation of endothelial cells from the arterial wall. There could be several potential injury sources such as chronic hyperlipidemia, chemical factors, immunological injury or mechanical injury. The endothelial denudation and exposure of the subjacent connective tissue would lead to the adhesion of platelets and platelet aggregation, releasing platelets constituents to the arterial wall. The platelets-derived factors along with lipoprotein and other plasma constituents would lead to migration and proliferation of VSMC in these sites of injury. The original response-to-injury hypothesis suffered several modifications. One of the first was that it was not necessary a denudating injury of the endothelium to initiate the development of an atherosclerotic lesion. Instead, an injury could trigger the process by producing endothelial dysfunction [23]. One of the initial manifestations of endothelial dysfunction would be the accumulation of lipid and lipoprotein particles under the endothelium due to increased endothelial permeability [23, 24]. Another feature of endothelial dysfunction would be an enhanced leukocyte and platelet adhesion. These cell types release cytokines and growth factors that promote an inflammatory response, with migration and proliferation of VSMC [25]. Recruitment of macrophages, and the accumulation of lipids inside of them, leads to the formation of foam cells, the hallmark of an initial atherosclerotic lesion [25]. Other two hypotheses are focused on the role of LDL as an initiator of the development of atherosclerotic lesions [19, 25]. Under certain conditions, non modified LDL particles could accumulate beneath the intima and initiate the process of atherogenesis, in which it could be of importance the interaction of apolipoprotein B-100 (apoB-100) and extracellular matrix proteoglycans [26]. This evidence supports the hypothesis of an accumulation of subendothelial LDL as an initial factor for atherogenesis. On the other hand, extensive

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evidence has been achieved sustaining the role of oxidized LDL in the pathogenesis of atherosclerosis. In brief, the oxidative modification hypothesis states that oxidative modification of LDL in atherosclerosis-prone areas leads to its uptake by macrophages and formation of foam cells [27, 28]. The role of LDL in the pathogenesis of atherosclerosis will be reviewed more extensively later in this chapter. The different hypotheses for the initiation of the development of an early atherosclerotic lesion are not mutually exclusive. On the contrary, evidence supporting each one of them shows that most probably they are complementary in explaining the early processes involved in atherogenesis. A more detailed review of the main actors involved in atherosclerosis pathophysiology is presented in the following paragraphs.

2.4. Role of the Different Components of the Atherogenesis Process 2.4.1. Low-Density Lipoproteins Low-density lipoproteins (LDL) are the main cholesterol carriers in plasma. The LDL particles are defined as lipoproteins with a density between 1.019-1.063 g/ml [29]. The main components of LDL are phospholipids, triglycerides, cholesteryl esters, unesterified cholesterol and apoB-100 protein. High plasmatic LDL levels have been considered a risk for the development of CVD [6]. Accordingly, accumulation of cholesterol in macrophages within the intima is considered an early feature of the atherogenic process [23]. Most macrophages internalize LDL through the classic LDL receptor [30]. However, this receptor is down-regulated when intracellular levels of cholesterol increase [30]. So, although macrophages can uptake LDL through this mechanism, the increase in intracellular cholesterol should down-regulate LDL receptors and this way prevent the accumulation of cholesterol and formation of foam cells. In fact, a diminished LDL receptor expression has been detected in foam cells of human atherosclerotic lesions [31]. In addition, patients suffering from homozygous familial hypercholesterolemia, a disease characterized by a total or near total absence of LDL receptors, develop severe atherosclerosis and macrophage derived foam cells are detectable [32]. This evidence suggests that a different mechanism of LDL uptake by macrophages must be present. Brown and Goldstein demonstrated that acetylation of LDL permitted its accumulation in macrophages, establishing the possibility that modified forms of LDL were involved in the pathogenesis of atherosclerosis [32]. It was demonstrated that this uptake is mediated by a specific scavenger receptor called the acetylLDL receptor [32, 33]. Also, it was demonstrated that isolated endothelial cells could modify LDL, allowing its uptake by macrophages, at least in part through the acetyl-LDL receptor [34]. Later, it was established that a modification associated with an increased uptake of LDL by macrophages was oxidation, proposing that free radicals should be involved in this process [35, 36]. Oxidation of LDL is the main oxidative modification in pathophysiology of atherosclerosis, and constitutes the basis of the original hypothesis of oxidative modification [25]. Oxidized lipids have been detected in atherosclerotic lesions of different stage of evolution. There is evidence regarding the presence of oxidized derivatives of fatty acids, such as hydroxyoctadecaenoic acids, and oxisterols, the oxidized derivatives of cholesterol

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[37]. Also, products of protein oxidation have been detected in human atherosclerotic plaques [38]. If oxidized LDL (oxLDL) is in fact participating in atherogenesis, it must be detected in atherosclerotic lesions in vivo. Experiments in rabbits and humans have shown that oxLDL is present in atherosclerotic lesions and absent in normal artery wall [39], and that LDL extracted from that lesions shares many biological properties with in vitro oxLDL [40]. Even more, evidence suggests that the uptake of lesion LDL occurs via scavenger receptors [40]. It is important to notice that oxLDL does not refer to a specific molecular species, but to a spectrum of heterogeneous forms of oxLDL [27]. An early form of oxLDL is referred to as minimally modified LDL (MM-LDL), which can still be recognized by LDL receptor and is not recognized by the scavenger receptor. It is not likely that LDL oxidation occurs in plasma, due to the abundance of antioxidants present in it [41]. Instead, the site of oxidation could be the arterial wall. There are several potential sources of ROS in the arterial wall that could participate in LDL oxidation. As it was explained in chapter 1, NADPH oxidase is a source of superoxide anion radical (O2-·). There is evidence of ROS production in the vascular wall through NADPH oxidase activity [42, 43]. Apart from macrophages, the presence of NADPH oxidase has been described in endothelial cells, VSMC and fibroblasts [44]. NADPH oxidase in vascular wall can increase its activity in response to several stimuli such as angiotensin II, platelet derived growth factor, TNF-α and, in endothelial cells, exposure to mechanical forces [44]. Xanthine oxidase (XO), another source of O2-·, has also been described in endothelial cells, with an increased activity in response to shear stress [45]. Another potential source of O2-· in the vascular wall is uncoupled endothelial nitric oxide synthase (later in this chapter). However, in consistence with its low reactivity, O2-· has a limited capacity to oxidize LDL [46]. Instead, it could be the precursor of more reactive oxidants [25]. Reactive nitrogen species (RNS), such as peroxynitrite resulting from the reaction of O2-· with nitric oxide (NO), can also be a source for oxidized LDL. Low-density lipoproteins isolated from human atherosclerotic lesions show higher levels of 3-nitrotyrosine in relation to plasma LDL of healthy subjects, which is consistent with LDL oxidation by RNS [47]. There is evidence supporting a possible role of lipoxygenases (LOs) in LDL oxidation. 15-lipooxygenase (15LO), an enzyme that is expressed in atherosclerotic lesions, is capable of oxidize LDL [48, 49]. In addition, inhibition of 15LO showed a protective effect against atherogenesis [50]. Myeloperoxidase (MPO) is another enzyme potentially involved in the pathogenesis of atherosclerosis [51]. Evidence shows that active MPO is present in atherosclerotic lesions [52]. In addition, 3-chlorotyrosine, a specific marker of MPOcatalyzed oxidation, is present in high levels in LDL isolated from atherosclerotic lesions [53]. Besides promoting foam cell generation, a number of other proatherogenic properties of oxLDL have been described and extensively reviewed [54, 55]. Oxidized LDL is chemoattractant for monocytes [56] and inhibits tissue macrophage motility [57]. Thus, oxLDL could initially recruit monocytes to the arterial wall. Later, after differentiation of the monocyte to a tissue macrophage, oxLDL would inhibit its migration, “trapping” it in the subendothelial space. Also, MM-LDL can increase the expression of monocyte chemoattractant protein-1 (MCP-1) [58] and macrophage colony-stimulating factor (MCSF) [59] in endothelial cells. Oxidized LDL increases the expression of Vascular Cell Adhesion Molecule-1 (VCAM-1) in endothelial cells [60], facilitating monocyte adhesion. In addition,

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oxLDL is mitogenic for macrophages [61] and VSMC [62]. Another effect of oxLDL is inhibition of NO induced vasodilation [63]. 2.4.2. Endothelium The endothelium is a single layer of endothelial cells that covers the luminal surface of vessels. Although originally it was thought to be only a structural barrier between the blood and the arterial wall, many other functions have been described, such as regulation of vascular tone, modulation of inflammation, regulation of vascular growth and modulation of platelet aggregation and coagulation [64]. Endothelium alterations have been implicated in the pathophysiology of atherosclerosis for a long time, as in the original response to injury hypothesis for atherogenesis, implicating a denudating endothelial injury. Endothelial dysfunction has a central role in atherogenesis, although the exact cellular and molecular processes implicated have not been completely elucidated. There is increasing evidence of a role of ROS in the developing of endothelial dysfunction. Regulation of vascular tone is one of the main functions of the endothelium (for more details see chapter 2). The hallmark of endothelial dysfunction is alteration of endothelium dependent vasodilation, which is mediated by NO. Nitric oxide is produced in endothelial cells by endothelial nitric oxide synthase (eNOS) from L-arginine (later in this chapter). Nitric oxide diffuses to VSMC where it activates the soluble guanylyl cyclase, increasing cyclic guanosine monophosphate (cGMP) production. The latter activates the cGMPdependent kinase I which in turn increases the opening probability of Ca2+-activated K+ channels, thereby inducing a hyperpolarization of the VSMC and inhibition of agonistinduced Ca2+ influx. This leads to VSMC relaxation and, in consequence, vasodilation. Apart from its role in the regulation of vascular tone, NO has a number of other functions in vascular homeostasis [65] including suppression of abnormal VSMC proliferation, control of hemostasis and fibrinolysis, and regulation of platelet and leukocyte interactions with the arterial wall. Therefore, impaired production or activity of NO leads to events or actions, such as vasoconstriction, platelet aggregation, VSMC proliferation and migration, and leukocyte adhesion, that promote atherosclerosis [66]. In consequence, any process leading to a decreased bioavailability of NO could be potentially proatherogenic. Assessment of endothelial dysfunction can be made through several techniques, such as the coronary response to the use of acetylcholine, flow mediated dilatation with brachial artery imaging, forearm plethysmography, finger-pulse plethysmography and pulse curve analysis [64, 67]. By this means, endothelial dysfunction has been detected in clinical conditions known to be risk factors for CVD such as hypercholesterolemia, hypertension, smoking, diabetes, and a positive family history of premature CVD [67, 68]. There is evidence supporting a role of ROS in reducing NO bioavailability. Endothelial dysfunction is associated with an increase production of ROS. Once formed, O2-· reacts with NO to form peroxynitrite. This reaction consumes NO, decreasing its bioavailability. Furthermore, under certain conditions eNOS can become uncoupled (later in this chapter), resulting in production of O2-·, which perpetuates the process [69]. Another stimulus for endothelium dependent vascular relaxation is shear stress. Shear stress is defined as the lateral force exerted on the endothelial cells by the passage of a semiviscous fluid over them. It is now well established that areas of the vasculature that

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experience unusual shear stress are particularly vulnerable to endothelial dysfunction, such as bifurcations, branch points and tortuous vessels [67]. Also, laminar flow has been proven to induce the expression of antioxidant response element-mediated genes that have a protective role against oxidative stress in endothelial cells [70]. There are other endothelium-derived vasodilators that could have a role in atherogenesis, including prostacyclin and bradykinin [66]. Prostacyclin acts synergistically with NO to inhibit platelet aggregation. Bradykinin stimulates the release of NO, prostacyclin, and endothelium-derived hyperpolarizing factor, another vasodilator, which contributes to inhibition of platelet aggregation. Bradykinin also stimulates the production of tissue plasminogen activator, and thus may play an important role in fibrinolysis. Activation of the endothelium by inflammatory stimuli results in the expression of a wide range of proteins that alter its function. Most notable among these are vascular cell-adhesion molecules. Several adhesion molecules are over-expressed on endothelial cells in atherosclerosis, such as intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin and VCAM-1 [71, 72, 73]. Intercellular adhesion molecule-1 binds to integrins present on all white cells. E-selectin binds to leucocytes expressing sialylated Lewis antigens, including neutrophils, monocytes, and memory T cell. Vascular cellular adhesion molecule-1 binds to a ligand present on lymphocytes, eosinophils, and monocytes. There is evidence of an antioxidant-sensitive mechanism involved in the expression of VCAM-1 in endothelial cells [74], which is concordant with an increased expression of this adhesion molecule after exposition to oxLDL [60]. 2.4.3. Inflammation Cells and Mediators The role of inflammation in atherosclerosis has been well established, leading to the concept that atherosclerosis in an inflammatory disease [24, 75]. Endothelial dysfunction leads to an increase of the expression of adhesion molecules in endothelial cells, such as VCAM-1, especially in regions with unusual shear stress (low average shear stress but high oscillatory shear stress). This leads to adherence of monocytes and T-lymphocytes. After adhesion, leukocytes migrate into the underlying intima in response to chemoattractant stimuli, including chemokines such as MCP-1. This inflammatory process stimulates migration and proliferation of VSMC that become intermixed with the area of inflammation to form an intermediate lesion. If inflammation continues, an increased number of monocytes and lymphocytes accumulate in the arterial wall, due to emigration from the blood and multiplication in the lesion, perpetuating the inflammation process [24].

Monocytes – Macrophages – Foam cells After adhesion to the endothelium and migration to the subendothelial space, monocytes mature into macrophages under the influence of MCSF, which is over-expressed in the inflamed intima [76]. Macrophage differentiation is a necessary step for atherosclerosis and is associated with up-regulation of pattern recognition receptors for innate immunity, including scavenger receptors and toll-like receptors (TLRs) [76]. As previously discussed, macrophages internalize oxLDL via scavenger receptors. The accumulation of cholesteryl esters in the cytoplasm leads to the formation of foam cells. Toll-like receptors bind certain ligands and initiate a signal cascade leading to macrophage activation [75]. Besides ligands

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such as bacterial toxins, TLRs can be activated by oxLDL and heat shock protein 60 (HSP60), which is highly expressed in atherosclerotic lesions of increasing severity [77]. Macrophage activation in atheroma leads to release of vasoactive molecules, ROS and metalloproteinases that may degrade matrix components. The loss of matrix components may subsequently lead to destabilization of plaques involving increased risk for plaque rupture and thrombosis.

T-Cells T-cells are present in atherosclerotic lesions, with a majority of CD4+ T-cells over CD8+ T-cells. Major histocompatibility complex (MHC) class II–expressing macrophages and dendritic cells can be detected close to these T cells. This implies a possible immune activation of T-cells in atherosclerotic lesions through processing and presentation of antigens by macrophages. Also, the atherosclerotic lesion contains cytokines that promote a T-helper 1 response, inducing activated T cells to differentiate into T-helper 1 effector cells. T-cell activation results in the secretion of cytokines, including interferon-γ and TNF- α and β that amplify the inflammatory response [24].

Markers of Inflammation in Atherosclerosis A crescent interest in establishing the utility of biomarkers for inflammation in atherosclerosis has developed in recent years. This is in part due to the potential utility of these markers for the assessment of early or advanced atherosclerosis in the clinical practice. Biomarkers include adhesion molecules such as VCAM-1; cytokines such as TNF-α, IL-1, and IL-18; proteases such as MMP-9; the messenger cytokine IL-6; platelet products including CD40L and myeloid-related protein 8/14; adipokines such as adiponectin; and acute phase reactants such as C-reactive protein (CRP), plasminogen activator inhibitor-1, and fibrinogen [78]. Among these, CRP has proved to be a useful marker, with a relatively easy and standardized detection. CRP is a strong and independent predictor for cardiovascular events, and can be used in addition to LDL levels for assessment of cardiovascular risk [79]. 2.4.4. Vascular Smooth Muscle Cells Vascular smooth muscle cells (VSMC) are the main cell type present in the media of arteries. It is well known that the central cellular feature of atherosclerosis is the accumulation of certain cell types, including VSMC, in the intima of arteries. Although the main localization of VSMC in vascular wall is the medial layer, it is possible to find VSMC in the intima of arteries in atherosclerosis-prone areas. These areas are characterized by intimal thickening that occurs mainly by accumulation of VSMC and proteoglycans secreted by them [18]. Given that atherosclerosis-prone areas are the preferential site for development of advanced atherosclerotic lesions, a possible role of VSMC in the initial development of atherosclerotic lesions has been proposed [80]. In advanced lesions VSMC migrate from the media and proliferate within the intima, forming part of the cellular component of atherosclerotic lesions. Vascular smooth muscle cells in the media of arteries have a contractile function, predominantly expressing proteins such as smooth muscle myosin heavy chain or smooth

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muscle actin. However, intimal VSMC associated with atherosclerotic lesions have a different phenotype, with lower expression of these proteins, higher proliferative index and greater synthetic capacity for extracellular matrix, proteases, and cytokines [81]. These two phenotypic states have been called contractile state and synthetic state, respectively [81]. Altered VSMC phenotype migrates and proliferates more readily and can synthesize more collagen. In addition, they express a greater proportion of VLDL, LDL and scavenger receptors allowing more efficient lipid uptake. Thus, VSMC can internalize and accumulate LDL and generate foam cells. Also, VSMC in atherosclerotic lesions express adhesion molecules VCAM-1 and ICAM-1, being able to bind and retain monocytes within the developing atherosclerotic lesion. Besides retaining monocytes, there is evidence supporting a protective effect of VSMC against monocyte apoptosis [80]. Vascular smooth muscle cells are capable of producing a wide range of cytokines, such as MCP-1, IL-8 and IFN-γ [82]. Among other functions, cytokines produced by VSMC attract and activate leukocytes, induce proliferation of VSMC, promote endothelial cell dysfunction and stimulate production of extracellular matrix components [80]. One of the main functions of VSMC in normal arteries and atherosclerotic lesions is the production of extracellular matrix (ECM). Extracellular matrix of healthy arteries consist mainly in type I and type III collagen, whereas atherosclerotic lesions contain mainly proteoglycans, type I collagen and fibronectin [80]. Apolipoprotein B-100 present in LDL can bind to proteoglycans, this way retaining LDL in the subendothelial space, where it can be oxidized [26]. Also, proteoglycans and fibronectin in ECM promote VSMC proliferation [80]. Predominantly in vitro studies have shown that rat and mouse VSMC can switch between the contractile and synthetic phenotypic states in response to a variety of atherogenic stimuli including extracellular matrix cytokines, shear stress, and lipids [80]. There is evidence supporting a role of ROS in the induction of a synthetic phenotype [83, 84] and of a contractile-differentiated phenotype [85, 86]. Despite this apparent contradiction, it has been established a role of ROS in promoting VSMC growth (hypertrophy and proliferation) and migration, expression and activation of metalloproteinases secreted by VSMC, and expression of inflammatory genes such as MCP-1 and interleukin-6 [87], further supporting a role of oxidative stress in the pathophysiology of atherosclerosis. 2.4.5. Platelets The presence of platelets in sites of endothelial injury has been known for a long time. Platelets have an important role in the thrombotic vascular occlusion following the erosion of a plaque. However, platelets may have a role also in the lesion formation [88]. There is evidence showing that platelets can adhere to non denudated endothelium in regions of activated endothelial cells [89]. Adhesion causes activation of platelets, involving the secretion of a variety of chemokines that potentiate the inflammatory response [88]. Also, platelets can promote monocyte transformation into foam cells [88]. The effects of ROS in platelet function are not clear since there are studies supporting a role of ROS in promoting platelet aggregation and studies that show an opposite effect. It appears that exposure to low levels of oxidants may promote aggregation and high levels of oxidants may inhibit it [90]. However, the role of oxidative stress in platelet function still has to be elucidated.

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2.4.6. Adventitia For a long time the adventitia was considered to have only a structural function, with no participation in pathologic vascular processes. However, in recent years novel functions of adventitia have been described, and its possible role in vascular homeostasis has been addressed. During atherosclerosis, infiltration of the adventitia by inflammatory cells can occur. The presence of NADPH oxidase activity in the adventitia allows this structure to be another source of ROS that can reduce NO bioavailability [91]. Also, oxidants can have a role in promoting fibroblast proliferation, this way participating in vascular remodeling [92]. 2.4.7. Urotensin II Urotensin II (U-II) is the most potent mammalian vasoconstrictor identified. Although an important role of U-II in hypertension pathophysiology has been established (see chapter 2), a variety of potential proatherogenic features have also been described [93, 94]. U-II is expressed in several cell types in atherosclerotic lesions, and its receptor is present in VSMC. U-II is chemotactic for monocytes and stimulates foam cell formation. Also, U-II promotes proliferation of endothelial cells and VSMC, induces the expression of NADPH oxidase and collagen 1 in VSMC, but decreases that of metalloproteinase-1. This way, U-II could be a link between hypertension and atherosclerosis. Accordingly, there is evidence that suggests an effect of U-II in atherosclerotic plaque formation and, moreover, U-II plasmatic level is an independent risk factor for the development of carotid atherosclerotic plaque in essential hypertensive patients, showing an association even stronger than that for some widely accepted risk factors, such as age or systolic blood pressure, among others [95].

2.5. Role of Oxidative Stress in the Pathogenesis of Atherosclerosis 2.5.1. Sources of ROS in the Vascular Wall There are several potential sources of ROS in the vascular wall, most of which have been proved to intervene in some degree in the atherogenesis process. This section exposes the evidence supporting the role of the main possible ROS sources in atherosclerosis.

2.5.1.1. NADPH Oxidase NADPH oxidase is a membrane-associated enzyme that catalyze the 1-electron reduction of oxygen using nicotinamide dinucleotide (NADH) or nicotinamide dinucleotide phosphate (NADPH) as the electron donor, this way generating O2-·. NADPH oxidase is considered to be the main source of ROS in the vascular wall [44]. This enzyme was first described in phagocytes and later in non-phagocytic cells such as the fibroblast [96]. In relation to atherosclerosis, there is evidence supporting its presence in VSMC, endothelial cells, adventitial fibroblasts and macrophages [97]. NADPH oxidase consists of several membranebound subunits (gp91, Nox and p22phox) and cytosolic subunits (p47phox, p67phox, p40phox and Rac2). In phagocytes gp91phox contains the putative binding sites for NADPH, heme and FAD, and together with p22phox, supports the flow of electrons from NADPH to O2. Although endothelial cells and adventitial fibroblasts express a gp91phox-containing NADPH oxidase similar to that originally identified in phagocytes, VSMC contain

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homologues of gp91phox, namely Nox1 and Nox4 [97]. NADPH oxidase activity has been linked to atherogenesis, since NADPH-derived ROS have a role in atherosclerotic lesion formation [98]. There are several stimuli that can increase NADPH activity, including angiotensin II, endothelin-1, U-II, growth factors such as thrombin and vascular endothelial growth factor (VEGF), cytokines such as TNF-α, metabolic factors such as increased glucose and insulin, oxidized lipids, oscillatory shear stress, hypoxia/reoxygenation, and nutrient deprivation [97, 99]. Angiotensin II, endothelin-1 and U-II are more related to hypertension pathophysiology, although they can be the pathophysiological link between hypertension and atherosclerosis (see chapter 2). Growth factors and cytokines are strongly expressed in atherosclerotic lesions, due to the characteristic inflammatory response previously referred in the chapter. As it was discussed previously, atherosclerosis prone areas are characterized for being exposed to altered blood flow which results in oscillatory shear stress instead of laminar shear stress. Laminar shear stress causes a transient increase in NADPH activity associated with an increase in superoxide dismutase (SOD) expression. In contrast, oscillatory shear stress causes a sustained increase of NADPH activity, with no increase in SOD levels [100]. This way, elevated ROS production by NADPH oxidase in these areas can have a role in initial lesion formation. Oxidized LDL can enhance the activity NADPH oxidase in leucocytes and endothelial cells through an induction of gp91phox expression, thereby potentiating the atherogenesis process [101].

2.5.1.2. Xanthine Oxidase Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are the two forms of xanthine oxidoreductase (XOR) enzyme. XDH is the original translational product of the XOR gene and XO derives from XDH through posttranslational modification [102]. Exposure of XDH to proteases leads to an irreversible proteolytic conversion to XO. Instead, a reversible conversion of XDH to XO can occur through oxidation of certain thiol groups [103]. XOR is generally recognized as the terminal enzyme of purine degradation in man, catalyzing the hydroxylation of hypoxanthine to xanthine and of xanthine to urate. Both XDH and XO catalyze the terminal steps in the metabolism of purines, but XDH binds NAD+ and XO does not. In consequence, XDH mediated reaction produces NADH, whereas XO transfers electrons directly to molecular oxygen, leading to the formation of O2-· [102]. Although the metabolic function of these enzymes has been known for a long time, a great interest in them has been developing in recent years, due to their capacity to generate ROS and their possible involvement in oxidative stress-related pathologies. In relation to atherosclerosis, XO is part of the multiple ROS sources potentially involved in atherogenesis. There is evidence supporting the presence of XO in atherosclerotic plaques [104]. Also, XO expression and activity is present in endothelial cells and increases in response to oscillatory shear stress. This effect requires the presence of NADPH oxidase [45]. Furthermore, in several experimental models the use of XO inhibitors attenuates endothelial dysfunction [105].

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2.5.1.3. Nitric Oxide Synthases Nitric oxide synthases (NOS) are a group of enzymes that catalyze reactions resulting in NO production. These enzymes are dimeric in their active form and for their activity require the presence of cofactors including tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and iron protoporphyrin IX [106]. Nitric oxide synthases catalyze the reaction of L-arginine, NADPH, and oxygen to NO, citrulline and NADP. There are three identified nitric oxide synthase isoforms: nNOS (type I, NOS-I or NOS-1), iNOS (type II, NOS-II or NOS-2) and eNOS (type III, NOS-III and NOS-3). nNOS is found in neuronal tissue, iNOS is an inducible isoform present in several tissues and eNOS is found in vascular endothelial cells. Inducible NOS is expressed in macrophages and smooth muscle cells in atherosclerotic lesions, promoting the formation of peroxynitrite [107, 108]. However, the role of iNOS in vascular pathology is variable and poorly defined in atherosclerosis [109]. There is evidence suggesting a possible protective effect of iNOS inhibition against atherosclerosis development [110]. The role of eNOS in atherogenesis has been largely studied. Physiologically, eNOS is regulated by caveolin-1 that binds to calmodulin and inhibits it. The binding of calcium to calmodulin-1 leads to displacement of caveolin-1 and activation of eNOS [66]. Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of eNOS. It has been reported an increase level of ADMA in hypercholesterolemic patients, establishing a potential role in atherogenesis [111]. Endothelial NOS uses BH4 as a cofactor for transfer of electrons from a heme group within the oxygenase domain to L-arginine to form L-citrulline and NO. If either BH4 or L-arginine is absent, eNOS switches to an uncoupled state, in which the electrons from heme reduce oxygen to form O2-·. Nitric oxide can react with O2-· to form peroxynitrite, thereby decreasing NO bioavailability and promoting atherogenesis. Infusion of BH4 can improve endothelial function in hypercholesterolemic patients [112]. Furthermore, oxidation of BH4 by peroxynitrite or by NADPH oxidase-derived ROS can lead to eNOS uncoupling [69, 113].

2.5.1.4. Mitochondria Mitochondria produce adenosine triphosphate (ATP) through a process called oxidative phosphorylation. Oxidative phosphorylation is the process by which ATP is formed as electrons are transferred from NADH or FADH2 (generated through the Krebs cycle) to molecular oxygen, through a series of electron transport carriers localized in the inner mitochondrial membrane. The electron transport carriers include: complex I (NADHubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), complex III (ubiquinol-cytochrome c reductase), and complex IV (cytochrome c oxidase). The majority of electrons transferred by the electron transport chain are coupled with the production of ATP. However, 1-2% of electrons leak out to form O2-· [114]. During mitochondrial oxidative phosphorylation under pathophysiological conditions the electron transport chain may become uncoupled, leading to increased O2-· production [115]. Furthermore, elevated production of ROS in mitochondria damages lipids, proteins, and mitochondrial DNA, increasing mitochondrial dysfunction. Of these, it is likely that mitochondrial DNA is the most sensitive to physiologically relevant ROS-mediated damage. In humans and apoE (-/-)

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mice, the extent of mitochondrial DNA damage correlates with atherosclerosis progression and can even precede atherosclerosis in young apoE (-/-) mice [116]. Mitochondrial dysfunction and increased mitochondrial ROS production can promote atherogenesis through several mechanisms including impairment of endothelial function and induction of VSMC proliferation or apoptosis [117].

2.5.1.5. Myeloperoxidase Myeloperoxidase (MPO) is a tetrameric heme protein present in monocytes, macrophages and neutrophils. This enzyme catalyzes the conversion of chloride to hypochlorous acid (HOCl), a potent chlorinating oxidant. Myeloperoxidase is the only human enzyme known to generate HOCl. Therefore, chlorinated biomolecules are considered specific markers of oxidation reactions catalyzed by MPO. There is evidence supporting a role of MPO in atherogenesis by promoting oxidation reactions in atherosclerotic tissues [51]. Active MPO is present in atherosclerotic lesions [52]. In addition, 3-chlorotyrosine, a “molecular fingerprint” of MPO-catalyzed oxidation, is present in high levels in isolated lesion LDL [53]. Also, MPO can generate in vivo RNS. Nitric oxide can autooxidize to nitrite (NO2–) and nitrate (NO3–). Nitrite and hydrogen peroxide can be used as substrates by MPO for generating RNS that can nitrate tyrosyl residues [118].

2.5.1.6. Lipoxygenases Lipoxygenases (LOs) comprise a family of enzymes capable of mediating selective lipid oxidation. These enzymes facilitate the stereospecific addition of oxygen to cis unsaturated fatty acids, resulting in the formation of hydroperoxy fatty acids. Classically, LOs are subdivided into the 5, 8, 12, and 15 LOs according to the positional specificity of their oxidation of the common fatty acid substrate, arachidonic acid [49]. There is evidence supporting a possible role of LOs in atherosclerosis development. The enzyme 15-lipooxygenase (15LO) is capable to oxidize LDL and is expressed in atherosclerotic lesions [48, 49]. It was found that 12/15-LO deficient mice show lower rates of VSMC growth, migration and ECM production, and reduced extension of atherosclerotic lesions [119, 120]. Accordingly, inhibition of 15LO attenuates atherosclerosis development in rabbits fed with a high cholesterol diet [50]. A growing interest in a potential role of 5lypooxigenase (5LO) in atherosclerosis has recently developed [121]. There is evidence of an increased density of 5LO expressing cells in the intima of progressively more severe lesions [122]. Also, LDL receptor-null mice show an important reduction of atherogenesis when they lack of a copy of the 5LO gene [123]. 2.5.2. Pathogenic Role of ROS in Atherosclerosis The effects of ROS in atherogenesis have been reviewed previously in this chapter in relation to each one of the components and processes involved in the development of atherosclerosis. As it was discussed, most of the actions of ROS that promote atherosclerosis occur through the generation of oxLDL and through a decreased NO bioavailability that leads to endothelial dysfunction. These include impaired vasodilation, expression of adhesion molecules and chemoattractant proteins, adhesion of monocytes, accumulation of oxidized lipoproteins, generation of foam cells, proliferation of VSMC and fibroblasts, and promotion

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of an inflammatory response. The effects of ROS in atherosclerosis pathogenesis are depicted in figure 3-1.

Figure 3-1. Scheme illustrating the hypothesis of the involvement of reactive oxygen species (ROS) in atherosclerosis. CVD, cardiovascular diseases; oxLDL, oxidized LDL.

3. Antioxidants in Atherosclerosis The great amount of evidence supporting a role of oxidative stress in the pathogenesis of atherosclerosis suggests that interventions consisting in the supplementation of antioxidants should have a protective effect against the development of atherosclerosis. A number of studies have evaluated the effect of a variety of antioxidants in atherosclerosis pathophysiology in vitro and in vivo. Most of these studies focus on vitamin E, because of its strong lipophilicity, whereas other natural or synthetic antioxidants, such as vitamin C, probucol and β-carotene, are supposed to play a minor role [124].

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Vitamin E is the collective name for molecules that exhibit the biological activity of αtocopherol. Both naturally occurring and synthetic forms of vitamin E are present. Naturally occurring forms of vitamin E include four tocopherols and four tocotrienols (α, β, γ and δ). Alpha-Tocopherol is the principal and most potent lipid-soluble antioxidant in plasma and LDL [125]. One LDL particle contains between 5 and 12 molecules of α-tocopherol. There are several lines of evidence suggesting a potential protective role of α-tocopherol against atherosclerosis [125, 126]. Supplementation with α-tocopherol results in a decreased susceptibility of LDL to oxidation [127]. Furthermore, in a placebo-controlled, randomized trial, it was established that the supplementation of α-tocopherol at a dose of 400 IU/d or more was effective in decrease LDL oxidation [128]. The use of α-tocopherol reduces the expression of adhesion molecules in endothelial cells, the adhesion of monocytes to endothelial cells, and the production of O2-· and IL-1β by monocytes [129, 130, 131]. Moreover, α-tocopherol prevents oxLDL-induced endothelial dysfunction in vitro and reduces endothelial dysfunction in vivo in hypercholesterolemic smokers [132, 133]. Alphatocopherol may have an effect in the inhibition of VSMC proliferation [134]. Also, αtocopherol can inhibit platelet adhesion [135]. Vitamin C (ascorbic acid) is a six carbon lactone that is synthesized from glucose in the liver of most mammalian species, but not in humans. Consequently, when humans do not ingest vitamin C in their diets, a deficiency state occurs that manifests as scurvy. Vitamin C is an electron donor and therefore a reducing agent. However, vitamin C itself is oxidized in the process, generating ascorbyl radical, which has a very low reactivity [136]. Ascorbic acid has an important capacity for preventing lipid peroxidation and LDL oxidation [137, 138]. The supplementation with ascorbic acid for 10 days was reported to reduce monocyte adhesiveness to endothelium in smokers [139]; however, another study showed no effect of ascorbic acid in monocyte adhesiveness [140], although supplementation in this case lasted for a shorter period of time (2 hours). Also, there is evidence supporting a beneficial role of ascorbic acid in endothelium-dependent vasodilation, this way preventing endothelial dysfunction [138]. In rabbits, supplementation with ascorbic acid has been proved to prevent hypercholesterolemia-induced atherosclerosis [141]. Carotenoids are the pigments responsible for the yellow to red color of some fruit and vegetables. The main carotenoids present in human diet are lycopene, lutein, α-carotene, βcarotene, β-cryptoxanthin, and zeaxanthin [142]. Various biological effects have been attributed to carotenoids, including antioxidant activity due to their capacity for scavenge ROS [143]. Supplementation with β-carotene has been shown to increase the content of this carotenoid in LDL and to inhibit endothelial cell-mediated LDL oxidation [144]. Moreover, high plasma carotenoid levels have been associated with a decreased risk for subsequent coronary heart disease events [145].

3.1. Clinical Trials Initial observational, epidemiological and case control studies showed conflicting results, but a possible protective effect of vitamin E was established [124]. Despite all the evidence supporting the antiatherogenic effects of antioxidant supplementation, clinical trials have

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failed to consistently prove a protective effect of antioxidants against atherosclerosis. A large number of randomized trials have been carried out, evaluating the effect of supplementing a single antioxidant or combination of them, including vitamin E, vitamin C and β-carotene. The results of these trials are contradictory. Although some of them attribute beneficial effects to antioxidants, most of them show no protective effect [124, 146, 147]. Even more, in some of these trials, an increase in mortality was reported in patients receiving antioxidant supplementation, which is also supported by meta-analyses, especially in relation to high doses of vitamin E [148, 149]. These conflicting results can be conditioned by a number of factors. It is important to take in notice the heterogeneity of the clinical groups studied, including patients with prior CVD, at risk of CVD, smokers, hipercholesterolemics and submitted to invasive interventions. There is also variation in the doses of antioxidants and the duration of the supplementation. Antioxidants may have a protective effect in initial phases of atherosclerosis, which could be lost in more advanced stages when the processes that perpetuate the development of the atherosclerotic lesion are established. This way, it could be expected a more consistent protective role of antioxidants in primary prevention. Therefore, further investigation in needed to consistently establish the efficacy of antioxidants in preventing atherosclerosis.

4. Conclusions and Perspectives There is a great body of evidence supporting a role of ROS in the pathogenesis of atherosclerosis. Most of the proatherogenic actions of an increased production of ROS are achieved through the production of oxLDL and through the consumption of NO that leads to endothelial dysfunction. Almost all of the processes involved in atherogenesis can be modulated by ROS, including attraction and adhesion of leucocytes, formation of foam cells, proliferation of several cell types and induction of an inflammatory response. There are several sources of ROS in the vascular wall, including enzymes such as NADPH oxidase and xanthine oxidase that could be potentially regulated in therapeutic interventions. All this evidence suggests that interventions consisting in supplementation of antioxidants should have a protective effect against atherosclerosis development. Consistently, there are several lines of evidence that show the multiple antiatherogenic effects of antioxidant supplementation. However, clinical trials have failed to prove a consistent protective effect. Although many clinical trials have been carried out, comparisons are difficult due to a great heterogeneity among studied populations and different supplementation duration and doses. It is possible that antioxidants could have a stronger protective effect in early stages of atherogenesis attenuating initial endothelial dysfunction, before the processes that perpetuate the formation of the atherosclerotic lesion are established. Further investigation is required to fully clarify the role of antioxidants supplementation in atherosclerosis.

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In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter IV

Postoperative Atrial Fibrillation

1

José Vinay1 and Ramón Rodrigo2

Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile. 2 Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile Supported by FONDECYT, grant 1070948

Abstract Atrial fibrillation is an arrhythmia occurring frequently within the first few days in 10% to 65% of patients after major cardiothoracic surgery (postoperative atrial fibrillation, POAF). It is associated with increased morbidity and mortality and longer, more expensive hospital stays. Despite the use of strategies to prevent POAF through the prophylactic use of agents such as β-adrenergic blockers, amiodarone, or others, a considerable percentage of the patients still presents the arrhythmia. The involvement of oxidative stress in the mechanism of POAF is supported by an increasing body of evidence indicating that the formation of reactive oxygen species (ROS) released following extracorporeal circulation are involved in the structural and functional myocardial impairment derived from the unavoidable ischemia–reperfusion cycle of this setting. ROS behave as intracellular messengers mediating pathological processes, such as inflammation, apoptosis and necrosis, thereby participating in the pathophysiology of POAF. Consequently, myocardial electrical and structural remodeling associates with the appearance of functional impairment consistent with alterations in electrical conduction. Therefore, it seems reasonable to assume that the reinforcement of the antioxidant defense system should protect the heart against functional alterations in the cardiac rhythm in this setting. Interestingly, exposure to low to moderate doses of ROS could trigger a cellular defensive response characterized by a prevailing effect of survival over apoptotic pathway, what should be considered a therapeutic target. The present chapter examines the molecular basis accounting for the contribution of oxidative stress to the development of POAF. In addition, it is presented the clinical and experimental evidence to support a new paradigm based in the prophylactic reinforcement of the antioxidant defense system toward reduction in the susceptibility of cardiomyocytes to ROS-induced injury.

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1. Introduction Atrial fibrillation (AF) represents the most common arrhythmia in clinical practice and is associated with poor clinical outcome. In the general population, it affects approximately 2.3 million people in the USA and increasing in fivefold the risk for stroke [1]. The efficacy of currently available treatments is sub-optimal. In turn, AF is the most common complication associated with coronary artery bypass graft surgery and other surgical procedures performed with extracorporeal circulation (postoperative atrial fibrillation, POAF). It occurs frequently within the first few days in 10% to 65% of patients after major cardiothoracic surgery and results in increased morbidity and length of hospital stay, having enormous cost implications in these patients. Its appearance increases with age and with the presence of known risk factors as arterial hypertension, coronary heart disease, diabetes mellitus and valve disease, among others. Management of POAF is often frustrating, and strategies vary widely from institution to institution. Despite all the efforts put into preventing POAF, including the use of β-blockers and amiodarone, a considerable percentage of the patients still presents the arrhythmia [2-4]. Its genesis and pathophysiology have been heavily studied in the last years, however the exact mechanisms behind POAF appearance and perpetuation, have not been clearly described so far. The involvement of oxidative stress in the mechanism of POAF is supported by an increasing body of evidence indicating that the formation of reactive oxygen species (ROS) released following extracorporeal circulation are involved in the structural and functional myocardial impairment derived from the ischemia–reperfusion cycle. Reactive oxygen species behave as intracellular messengers mediating pathological processes, such as inflammation, apoptosis and necrosis, likely followed by fibrosis, thereby participating in the pathophysiology of POAF. Consequently, myocardial electrical and structural remodeling associates with the appearance of functional impairment consistent with alterations in electrical conduction. The lack of efficient and relative risk-free treatments has supported the search for novel drugs or agents that can cover the needs of these patients. In this context, a relative new line of study that associates POAF to oxidative stress is arising [5-7]. Numerous studies have suggested the pathophysiological link between POAF and oxidative stress, being the latter substantially present in the unavoidable ischemia/reperfusion cycle of this setting, thus giving rise to the involvement of ROS as pathogenic factors of the functional and structural impairment known to occur. The new paradigm that puts ROS as main players in the pathogenesis of POAF also supports the concept that pharmacological treatments that could intercept the mechanisms behind ROS production, propagation or action, at the same time could prevent or potentially treat this rhythm disorder. In this group, it can be found drugs with intrinsic antioxidant power as vitamin C, vitamin E, N-acetylcysteine (NAC) and statins; all which gather biological and pharmacological properties that make them excellent candidates in the treatment of this pathology [8-11]. Nevertheless, it should also be considered that agents causing up-regulation of antioxidant enzymes, such as catalase, superoxide dismutase and glutathione peroxidase, would be expected to have a beneficial effect against the deleterious action of ROS on cardiomyocyte function. The role of oxidative stress in the pathogenesis of AF and POAF, and their possible attenuation by antioxidants will be analyzed in the following sections.

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2. Pathophysiology Normal heart electrophysiology requires the correct function of the four intrinsic properties of cardiac cells: excitability, conductivity, contractility and automatism. Both structural and/or functional impairment of any of these properties could lead to heart disease, especially to a rhythm disorder. In healthy individuals, the cells with the higher intrinsic frequency of depolarization are located in the right atria (the heart pacemaker), and are denominated as a whole as sinus node. In this specific location, the cardiac depolarization wave starts. Firstly, the depolarization wave travels to the right and left atria, leading to their contraction. At the same time, other depolarization wave is travelling to the atrioventricular node, finally reaching the Hiss-Purkinge network and depolarizing both ventricles, leading to their contraction and the posterior ejection of blood into both aorta and pulmonary arteries. The most common disease, related to heart electric conduction, is AF [1]. Clinically, this tachyarrhythmia presents with a cardiac frequency over 90 pulsations per minute, involving the co-existence of two pivotal pathogenic events: increased cardiac cell automatism and the presence of reentry foci (the level of influence of each one, will determine the type of AF) [12-15]. Increased automatism, represented by the generation of rapidly discharging foci, means that cardiomyocytes located in different areas of the atria that should be overshadowed by the heart pacemaker, which intrinsically has a higher depolarization rate, start to act like new pacemakers, thus leading to an incorrect electric conduction and therefore a poor atrial contraction. Increased automatism is particularly clear in patients with focal AF, which have ectopic rapidly discharging foci usually near the pulmonary veins [12, 13]. This high frequency depolarization wave can not be properly conducted through the atria tissue and could convert into extra systoles, causing the atrium to fibrillate. On the other hand, the existence of reentry foci means that those new impulses generated in the context of increased cardiac cell automatism, are perpetuated by new re-entry wavelets. This is usually the consequence of a chronic heart injury, such as hypertension, coronary or valve disease, leading to dilation and fibrosis, all of which alter the electrophysiological properties of the heart, thus helping to perpetuate AF [16]. For the heart to experiment those pathogenic events has to suffer a constant stress for a long time, leading to a remodeling, which has two faces. For one hand the atria undergoes and electric remodeling based on electrophysiological changes, like shortening of the refractory period, decrease in the action potential and activation of cardiomyocytes automatism properties, thereby contributing to the genesis of ectopic discharging foci and reentry wavelets [5, 17, 18]. On the other hand, the atria suffers a structural remodeling, based on atrial dilation and fibrosis, secondary to the activation of different inflammatory and profibrotic mediators as angiotensin II, transforming growth factor β1 and tumor necrosis factor alpha1; leading to changes in heart electric conduction properties, thus helping to the perpetuation and generation of new re-entry foci [19, 21]. Therefore, both electric and structural remodeling generates the two main events necessary to the genesis and perpetuation of AF: ectopic automatism and re-entry wavelets. However, the mechanisms accounting AF genesis and perpetuation are quite different when analyzing different subpopulations. For example, POAF is the result of several heart injuries that co-exist in the post-operative state. Among them, the increase in the adrenergic

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tone [22], the activation of the renin-angiotensin system [23, 24], inflammation, ischemia and preoperative injuries associated with cardiac diseases (ventricular hypertrophy, atrial dilation and fibrosis, hypertension and necrotic zones secondary to atherosclerotic events) are the most important. The increase in the adrenergic tone and the activation of the reninangiotensin system deserve special mention because of the multiple mechanisms accounting for heart damage. Angiotensin II acts on vascular smooth muscle cells leading to general vasoconstriction; at the same time aldosterone generates renal sodium retention. Those two events, acting together, lead to hypertension, which increases heart oxygen consumption and ventricular stress, which in the long-term could end with cardiomyocyte hypertrophy and ventricular failure. Also, angiotensin II exerts an action directly over the ventricular tissue, causing extracellular matrix remodeling, which is reflected in atria dilation, hypertrophy and/or fibrosis [23, 24]. The increase in the adrenergic tone is a pivotal link in the pathophysiological chain accounting for AF genesis and perpetuation. The existence of ectopic rapidly discharging foci in many individuals can go unnoticed. However, when other risk factors are present, in this case, increased adrenergic tone, normal heart electric conduction is impaired: new discharging foci appear and the first ones perpetuate. This leads to asynchronous atrial electrical activation, and therefore to loss of atrial contractility. At the same time, new reentry foci emerge, generating multiple re-entrant wavelets that help to the perpetuation of the conduction abnormality [12, 13].

2.1. Oxidative Stress Oxidative stress has been found to play a crucial role in the pathogenesis of several cardiovascular diseases. One of the most studied has been AF and particularly POAF [6, 7, 25]. Following cardiac surgery, and especially with extracorporeal circulation, ischemic phenomena and posterior reperfusion are mandatory. This leads to the synthesis of high concentration of ROS, which could impair the normal operation of several physiological processes in the organism [26]. Before being determined the specific molecular pathways through which ROS exert their actions, the first evidences accounting for this hypothesis were the high levels of serum myocardial oxidation biomarkers (peroxide, derivatives of reactive oxidative metabolites of oxygen and/or nitrogen) in AF presenting patients in relation to healthy individuals [7, 26, 27]. There is also evidence for oxidative injury in atrial tissues from AF patients [26]. On this line, it was found that patients developing POAF had increased levels and expression of NADPH subunit Nox2 and in NOX-derived superoxide generation [28, 29]. Together with NADPH oxidase, it has been found that other pro-oxidative enzymes are in higher activity in the context of POAF; this is the case of xanthine oxidase and uncoupled nitric oxide synthase (NOS) [28]. Hence, it has been objectified that oxidative stress is present in this setting. ROS production is far from being a simple process; on the contrary, it is a complex mechanism involving pre- and post-transcriptional regulation. In the next paragraphs it will be presented the experimental data and theoretical bases of the different targets of ROS action, accounting for AF and POAF production and perpetuation.

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2.1.1. Electric Remodeling Electric remodeling is one of the most important mechanisms by which ROS disturbs the normal electric conduction of the heart. Fibrillating atria is characterized by a diminished action potential and effective refractory period, due to changes in several currents that normally maintain the cardiomyocytes electric potential [17]. Between those currents, the Ltype voltage-gated Ca2+ current has been found to be the principal target of ROS action. This current has been found to be diminished in cells extracted of fibrillating atria, as a result of cardiomyocytes calcium overloading [5, 17]. To completely understand how ROS produce calcium overloading and therefore diminish L-type voltage-gated Ca2+ current it is important to take into account the normal cardiomyocyte calcium homeostasis. Calcium influx into the cytosolic space is mediated largely by the ryanodine receptor Ca2+ channel (RyRC), which moves calcium between the sarcoplasmic reticulum (SR) into the cytosol. Physiologically, RyRC release calcium as a response to the arrival of an action potential to the cardiac cell [30]. However, experiments using canine SR vesicles demonstrated the existence of a superoxide activated calcium releases from RyRC [31]. Hence, ischemia-reperfusion dependent ROS could activate the RyRC and produce calcium overloading. As a consequence, L-type current is reduced, thus leading to the electrophysiological changes, like shortening of the refractory period, involved in the initiation and perpetuation of POAF. Finally, it has to be mentioned the effect of ROS in the disruption of cardiomyocytes connexins. Connexins are a set of proteins assembled between two adjacent cardiac cells, forming the structure known as gap junction. This structure participates in the efficient and rapid conduction of the electric potential through the cardiac tissue. Disruption of connexin 43 has been correlated with increased propensity for tachyarrhythmias [32]. Under conditions of oxidative stress, following an ischemia/reperfusion cycle, increased ROS directly interact with the connexins, particularly with connexin 43, thereby disrupting its organization, leading to electrical remodeling and therefore to propensity to present AF [33,34]. The exact molecular mechanism by which ROS disrupt normal connexin distribution has not been completely identified. However, the ROS-mediated activation of protein kinase C gamma, unique isoform present only in neural and optic tissue, leads to the phosphorylation and posterior disassembly of connexin 43 [35]. 2.1.2. Muscle Mechanical Impairment Myofibrillar creatine kinase (MM-CK) has a crucial role in muscle energetic metabolism. MM-CK buffers ATP concentration during the turnover happening in muscle contraction and relaxation. ROS may be involved in MM-CK oxidation, and therefore in the reduction of its activity as seen in AF developing patients undergoing Maze procedure in relation to non-AF presenting patients undergoing cardiac surgery [26, 36]. These findings are of vital importance, because set the precedent that atrial fibrillation does not reflect exclusively an electrophysiological issue, but on the contrary it is the result of several co-existent elements, among which it could be found the mechanical muscle impairment, as a result of a energetic deficit that may be involved in the lack of synchronously atrial contraction and therefore help to perpetuate fibrillation.

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2.1.3. Mitochondrial Damage Mitochondria are the energetic cellular organelles, so any injury that may suffer could cause cellular energetic impairment that could lead, depending on the intensity of the injury, to apoptosis or different levels of cellular damage. Mitochondrial DNA (mtDNA) is a potential target for ROS to produce oxidative damage. Through quantitative PCR technique, it was shown that the mtDNA of AF patients had more deletions than the mtDNA of patients in sinus rhythm. This is based on the high concentration of oxidative DNA damage products found in the first ones [37]. The damage done at this level has to be put in the context of oxidative stress perpetuation. For this, is important to remember that ROS production is frequently a reflection of cellular energetic impairment due to hypoxia and/or intracellular organelle damage, among others, where as there is a lack of oxygen, the electron transport chain cannot function correctly and therefore ROS are heavily produced. The damage done to mitochondria, involved in the majority of the cell energetic processes, may lead to an increment of ROS production rates, and those ROS at the same time will perform oxidative damage on mtDNA, impairing mitochondrial function, and therefore closing the vicious cycle. In brief, mtDNA ROS-mediated damage performs a positive feedback on ROS production that, at the same time, perpetuates mitochondrial damage and ROS synthesis. 2.1.4. Transcriptional Modulation It is of great importance to be acquainted with the notion that ROS not only exert their actions by directly modifying the constitution of different organic molecules. In addition, ROS also are involved in the regulation of several genes expression. These are ROS-sensitive genes, as they respond to changes in the cellular oxidative state. Many trials have reported effects of ROS in redox sensitive gene expression. It has been described that the presence of oxidative stress in AF patients results in changes accounting for a shift from the synthesis of antioxidant proteins to pro-oxidant ones [38]. Trials studying patients undergoing coronary artery bypass grafting or valve procedure described significant differences in genomic response between the patients that presented POAF and the ones that maintained in sinus rhythm; the first also showing the highest oxidative stress related parameters [27]. Microarray studies have demonstrated the existence of genes specifically associated with both AF and sinus rhythm patients. Among the first, the authors described molecules related to inflammation and different ion channels [39]. In total, there are described over 100 genes modulated between AF and sinus rhythm specific genes, and it is plausible to believe that ROS are involved in the modulation cascade of the AF intracellular transcriptional events. It is important to highlight the ROS-mediated activation of transcriptional factors, such as NF-κB and AP-1. These factors stimulate the transcription of several protein mediators, like proinflammatory cytokines that activate several cell death pathways, through apoptosis and/or necrosis [40]. The heart tissue, being subjected to this chronic injury, responds with a pathological regeneration, which contributes with the electric and structural remodeling of the tissue. For many years the studies involving oxidative stress and ROS have focused in the direct mechanism by which ROS altered the structure and function of different cell molecules. In the last years the study of the genetic mechanism by which ROS are involved have opened a completely new line of study that could help to intercept the different molecular locations

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where ROS produce damage, and therefore the new locations where novel therapeutic tools may help to treat or prevent different types of oxidative stress-mediated disorders. More studies to analyze the function of these genes are still lacking to date. 2.1.5. Oxidative Stress and Inflammation Nexus The pathogenesis of AF and POAF is a complex web of events that involves the activation of many processes, being inflammation one of the majors [24, 51]. Thus, it was shown that white cell count [42], as well as the levels of C- reactive protein is more elevated in the postoperative period, at day 2, in patients that experience POAF than in those that do not [43]. The role of cytokines, chemokines, leukocytes and acute-phase proteins, like highsensitivity C-reactive protein in the pathogenesis of POAF has been reported in several studies [44, 45]. At first sight, it could be thought that inflammation does not have anything in common with oxidative stress and that its origin, mediators and targets are completely specific for each process. But the reality cannot be more different from this concept, since oxidative stress, ROS and inflammation are a continuous that is very difficult to dissect. These phenomena have important molecular bridges that are activated in presence of ROS [46], leading to the activation of multiple mechanisms that end up causing heart tissue remodeling and therefore enhancing the susceptibility to present rhythm disorders. Among those molecules, the most studied has been the transcriptional factor NF-κB, a factor that responds to changes of the cellular oxidative state, ischemia-reperfusion and inflammatory molecules [47]. When NF-κB is activated, for example in presence of ROS, by phosphorylation of its inhibitory cofactor, it bonds to a DNA response element and promotes the transcription of genes involved in inflammatory and pro-fibrotic response, interleukin-6, transforming growth factor beta and tumor necrosis factor alpha [48]. Those molecules act in various tissues, but particularly at the heart, producing extracellular matrix remodeling and fibrosis (structural remodeling), which changes the electrophysiological properties of the heart making it susceptible to generate new re-entry foci and therefore perpetuate conduction abnormalities generated from rapidly discharging foci. Several studies have associated NF-κB activation with cardiac dysfunction, ventricular hypertrophy and maladaptive cardiac growth [19]. Different inflammation markers have been found in increased levels in serum and atria biopsies of AF and POAF patients [5, 7, 24, 44]. Therefore it is reasonable to assume that oxidative stress and inflammation response act in a synergic way in the underlying mechanisms of AF and POAF, giving the foundation for studies involving anti-inflammatory AF therapy. A schematic representation of the events associated with AF genesis and perpetuation is presented in figure 4-1.

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Figure 4-1. Schematic diagram illustrating a hypothesis based on the main contributory factors involved in the genesis and perpetuation of atrial fibrillation. AF, atrial fibrillation; NF-κB, nuclear factor kappaB; ROS, reactive oxygen species; AT II, angiotensin II; IL-6, interleukin-6; hsCRP, high sensitive C-reactive protein; RyRc, ryanodine receptor Ca2+ channel; ERP, effective refractory period; MM-CK, myofibrillar creatine kinase.

3. Prevention of Postoperative Atrial Fibrillation by Antioxidants Based on the numerous evidence supporting the hypothesis that oxidative stress is a cornerstone in the pathogenesis underlying POAF it could be noted that the use of antioxidants as therapeutic tools appears to be a rational line of study. Substances with

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antioxidant properties such as statins, N-acetylcysteine, and specially vitamins C and E have probed to be efficient in not only decreasing the serum oxidative levels in patients undergoing cardiac surgery, but also diminishing the occurrence of POAF [10,49-52]. Furthermore it has been hypothesized that one of the mechanisms whereby classic anti-AF drugs act is related with the ability to scavenge ROS and protect against membrane lipid peroxidation [53]. However, with all the evidence gathered to date, vitamin C (ascorbate) and vitamin E (α-tocopherol) highlight among other antioxidants, gathering several biochemical and empiric evidence that makes them excellent candidates to be used in the treatment and/or prevention of AF and POAF. The available evidence supporting the use of each one of these agents will be heavily discussed below.

3.1. Statins Statin drugs have both antioxidant and anti-inflammatory properties and several studies argue that their cardiovascular protection ability is part of their pleiotropic effect and goes beyond the cholesterol lowering effect alone [54,55]. The pleiotropic effect involves an improvement of endothelial function, enhancement in the stability of atherosclerotic plaques, decrease of oxidative stress and inflammation, and inhibition of the thrombogenic response. With regard to POAF, it has been observed that preoperative statins diminished the incidence of POAF in patients undergoing cardiac surgery [10, 54, 55]. Furthermore, statins attenuates AF promotion by atrial tachycardia in dogs [58]; and has been reported a decreased in the latter appearance of AF in patients undergoing electric cardioversion [56,57]. Recently, a meta-analysis of over 30000 patients showed that POAF incidence when using preoperative statins diminished from 29.3% (in the no-statins groups) to 24.9% [11]. All evidence point towards statins capacity to prevent AF and POAF, based on their antioxidant effect.

3.2. N-Acetylcysteine N-acetylcysteine (NAC) is a drug used for multiple purposes in clinical medicine [59]. In the last years, its antioxidant ability has called attention, leading to subsequent research in the prevention and/or treatment of AF and POAF. A prospective, randomized, placebo-controlled trial was conducted to study the potential anti-arrhythmic effect of NAC [10]. In this study of 115 patients undergoing coronary artery bypass and/or valve surgery, 58 patients received pre-operative NAC and 57 patients received placebo (both groups received also standard medical therapy, including β-blockers). The results showed that POAF incidence was 5.2% in the NAC group and 21.1% in the placebo group. These data demonstrated that an antioxidant agent, such as NAC, used in combination with classic anti-arrhythmic drugs, could contribute to prevent the appearance of arrhythmias like POAF. More studies using NAC are still lacking to assess the actual potential of this agent.

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3.3. Biological Properties and Synergism of Vitamins C and E Vitamin C and vitamin E are essential antioxidants that perform their roles in different cell locations, while the first one acts in water-soluble components the second one does it in lipid-soluble zones (mainly biological membranes or lipoproteins). Therefore all cell components could be protected against oxidative damage when both vitamins are used together [60, 61]. The most studied mechanism whereby they act is partly based on their property to directly reduce ROS. In addition to its ROS scavenging functions, these two antioxidants exerts their action in a synergistic way: when α-tocopherol losses and electron and is left like α-tocopheroxyl radical, vitamin C reduces it, so it can maintain its antioxidant properties [51, 52]. In the absence of efficient reducers, vitamin E cannot be recycled into its antioxidant form, leading to the formation of tocopheryl quinone, molecule that could compete in mitochondrial respiratory chain reactions. Hence, the therapeutic strategy presented in this chapter is based in the associated administration of both ascorbate and αtocopherol, ensuring the efficient recycling of vitamin E radicals [62, 63].

3.4. Endothelial Modulation Besides their ROS scavenger actions, vitamins C and E exert a complex modulation of numerous enzymes involved in ROS production, endothelial dysfunction, platelet aggregation and smooth muscle cell tone [27, 64, 65]. The four most important mechanisms in which antioxidant vitamins modulates the endothelial function are: NADPH down-regulation, and up-regulation of eNOS, phospholipase A2 and antioxidant enzymes. NADPH oxidase, the most important superoxide source in the cardiovascular system, can be directly down-regulated by vitamins C and E. The mechanism behind this effect has not been completely elucidated. It has been reported that ascorbate and α-tocopherol could be involved in NADPH oxidase transcriptional and post-transcriptional modulation. Moreover, studies describing a possibly direct effect to the NADPH oxidase synthesis have also been presented. Vitamin E could be involved in inhibiting the enzyme subunits aggregation, based in the location (membranous organelle) in which this process takes place [66]. In the presence of oxidative stress, eNOS is mostly in its uncoupled form, participating in superoxide production and NO synthesis impairment, all which leads to endothelial dysfunction. In this context, antioxidant vitamins have shown to increase eNOS activity, by enhancing the intracellular availability of tetrahydrobiopterin (one of its cofactors) and by inhibiting the p47phox subunit expression. Therefore, ascorbate and α-tocopherol increase NO synthesis, reduce ROS formation and contribute to the vascular tone regulation [66-69]. In relation to antioxidant enzymes up-regulation, some studies have demonstrated a positive correlation between antioxidant vitamins and antioxidant enzymes activity, particularly SOD. The mechanisms underlying these findings are not well elucidated, but it is plausible to hypothesize the existence of transcriptional and post-transcriptional events involved in the up-regulation of those antioxidant enzymes [65]. Finally, vitamin E also modulates the vascular prostanoid synthesis by up-regulating phospholipase A2 expression and therefore arachidonic acid (precursor of prostanoids, and

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leucotrienes) release; and down-regulating cyclooxygenase-2 expression. The final result is a net increase in vasodilator prostanoids, which contribute to the regulation of the vascular tone [70]. A schema with the proposed effect of antioxidant agents in the reinforcement of the myocardial antioxidant defense system is depicted in figure 4-2.

Figure 4-2. Schema with the proposed paradigm for the effect of antioxidant agents in the reinforcement of the myocardial antioxidant defense system. NAD(P)H oxidase, reduced nicotine adenine dinucleotide phosphate oxidase; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; NAC, Nacetylcysteine.

3.5. Empiric Evidence Supporting the Beneficial Effects of Vitamins C and E 3.5.1. In Vitro Studies and Animal Trials Vitamins C and E have demonstrated intrinsic abilities in preventing cell apoptosis, necrosis and cardiac dysfunction. Several studies have established their role in preventing

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oxidative damage in in vitro cardiomyocytes. Thus, when isolated cardiomyocytes were exposed to singlet oxygen oxidative damage, which lead to irreversible hypercontracture of 95% of the cells, the pre-treatment with vitamins C and E reduced the hypercontracture percentage in direct correlation with the vitamin concentration. This effect was enhanced when using both vitamins simultaneously [71]. Cardiomyocyte apoptosis has also been prevented by administration of antioxidant vitamins, which was also correlated with the diminution of oxidative stress biomarkers [72, 73]. Electrophysiological changes, secondary to hypoxia mediated injuries in guinea pigs cardiomyocytes, were prevented upon ascorbate administration. Vitamin C generated an important attenuation in the hypoxia related sodium current disturbance [74]. There also exist available data supporting vitamins anti-arrhythmic specific properties. In isolated rat hearts undergoing ischemia-reperfusion injuries, vitamin E showed an effective prevention in the appearance of reperfusion arrhythmias [8]. Furthermore, several animal models have been used to assess the favorable effects of vitamin C and E in the prevention of necrosis-apoptosis events, oxidative damage, calcium overloading and cardiac dysfunction [75-77]. Antioxidant vitamin anti-necrosis properties were established considering that cardiomyocytes necrosis events, of rats submitted to stimulation of myocardial infarction was prevented by the administration of vitamins C and E [73]. Myocardium fibrosis and remodeling play an important role in AF and POAF genesis and perpetuation. In this regard, α-tocopherol has shown important effects in preventing cardiac remodeling in spontaneously hypertensive rats, based on the inhibition of cardiomyocyte hypertrophy [78]. In addition, cardiac dysfunction attenuation through vitamin administration was demonstrated using rabbit models. Antioxidant vitamins were administered after pacing-induced cardiac dysfunction; subsequently, it was found a decrease in myocardial oxidation biomarkers, an attenuation of the pacing-induced cardiac dysfunction and a reduction in cardiomyocyte necrosis biomarkers [70, 72]. Both in vitro and animal trials have helped to understand the actual potential that antioxidant vitamins could have in preventing AF. Although the molecular basis and the in vitro evidence that supports their use in the prevention and/or treatment AF and POAF has been accumulating over the last years (evaluating cardiomyocyte contractility, apoptosis, electrophysiology, and isolated hearts arrhythmia appearance). It is necessary to gather all efforts in performing clinical trials, based importantly in the innocuousness of ascorbate and α-tocopherol administration. 3.5.2. Clinical Trials to Prevent Postoperative Atrial Fibrillation Antioxidant vitamins and AF related clinical trials have not been heavily studied. The advances made in this direction are presented in the following paragraphs. One of the most paradigmatic studies involving ascorbate anti-arrhythmic properties was conducted to test not only the effects of vitamin C supplementation in POAF incidence, but also to assess the biochemical changes in oxidative and electric status after canine atrial pacing. In the first part, 43 patients subjected to coronary artery bypass were given 2 g of vitamin C the day before the surgery, followed by 500 mg until the fifth post-operative day. The POAF incidence in the ascorbate treated group was 16% v/s 35% in the control group. In

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the other part of the study, eleven dogs were subjected to rapid atrial pacing, which led to shortening of the effective refractory period (ERP), associated with accumulation of 3nitrotyrosine, a peroxynitrite oxidative marker, and decreased levels of ascorbate compared with non paced controls. Ascorbate treatment attenuated the ERP shortening and diminished the 3-nitrotyrosine concentration found after atrial pacing [9]. This study showed, on the one hand that antioxidant vitamins could decrease the incidence of POAF, and on the other hand showed that this effect could be a reflection of a stabilization of the electrophysiological properties of the heart, that are impaired in individuals presenting this arrhythmia. It should be remembered that one of the mediators accounting for the shortening of the refractory period is superoxide (which leads to the cardiomyocytes calcium overloading), therefore it is plausible to occur an attenuation of the shortening ERP in presence of antioxidants. The effects of ascorbate administration in relation to AF have been tested under different contexts. A trial studied 44 patients subjected to electrical cardioversion of persistent AF, all received standard treatment, but one group received vitamin C during 7 days, while the other received only ordinary drugs. Within a week, AF recurred in 4.5% of the ascorbate treated group and in 36% of the control group [79]. In addition, antioxidant vitamins have been studied in the prevention of post-thrombolysis AF; comparing two groups subjected to therapeutic alteplase thrombolysis, one receiving antioxidant vitamins and the other placebo, the results showed that the first one developed AF after reperfusion in 6% while the placebo group presented the arrhythmia in 44% [80]. Post-thrombolysis AF is the gold standard example for ROS induced AF. Before the re-vascularization procedure, the heart tissue was experimenting high levels of hypoxia; whereas after the administration of the thrombolytic therapy (in this case alteplase), the heart suffered an acute restoration of blood and oxygen, which lead to calcium overloading of cardiomyocytes, activation of cell death pathways and production of high levels of ROS, phenomenon known as ischemia/reperfusion. Through all those mechanisms, electric properties of the heart were deregulated, and heart tissue was incapacitated to correctly conduct the electric impulses and therefore AF was observed. Recently, it was shown that oral vitamin C in association with β-blockers was more effective in preventing POAF than β-blockers alone. This study consisted in 100 patients undergoing coronary artery bypass grafting, separated in a β-blockers group and a βblockers/ascorbate group, which received 2 g of ascorbic acid on the night before the surgery and 2 g daily for 5 days after surgery. The POAF incidence was 4% in ascorbate group and 26% in the control group [81]. Consequently, antioxidant vitamins not only have shown favorable anti-arrhythmogenic results compared with non-vitamin patients, but also with patients receiving classical anti-AF drug treatment. This concept has major relevance, as the study for alternative therapeutic tools was originated because of the lack of effective and riskfree treatment for AF. The future task is to continue testing antioxidant therapies under different protocols and contexts, to assess their real potential in preventing and/or treating AF and POAF.

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4. Conclusions and Perspectives Oxidative stress plays a key role in the development of atrial fibrillation (AF), the most common arrhythmia in the general population. Increased production of reactive oxygen species (ROS) in myocardial tissue occurs in the unavoidable ischemia-reperfusion cycle produced during cardiac surgery with extracorporeal circulation. On this line, it is also conceivable the contribution of ROS in the development of postoperative AF (POAF), a frequent complication associated with poor clinical outcome of patients. Therefore, the deleterious effect of ROS could be counteracted by a reinforcement of the myocardial antioxidant defense system, involving either its non-enzymatic or enzymatic components. This paradigm lead to consider the administration of antioxidants, before cardiac surgery, in order to diminish the vulnerability of the heart to present the arrhythmia, thus avoiding, or at least mitigating the electrical and structural tissue remodeling caused by ROS exposure. Accordingly, evidence from both experimental studies and clinical trial has given a clue to the potential role of antioxidants, particularly vitamins C and E, in diminishing the incidence of POAF. After an initial approximation to the subject, the preconceived concept that ROS main function is to destroy and alter biological molecules, such as membranes and proteins, is replaced with the new paradigm that presents ROS as multi-tasking mediators that perform its actions through multiple mechanisms such as pre and post-transcriptional modulation, electric and structural tissue remodeling, energetic impairment and activation of parallel processes like inflammation. All those mechanisms together account for the high rates of AF developing in patients that are submitted to procedures that are intrinsically linked with ROS production, such as cardiac surgery, extracorporeal circulation and re-vascularization procedures. The fact that oxidative stress has been found to play an essential role in the pathological events related to this rhythm disorder, it is crucial to the future of therapeutic research in this field. Available pharmacologic treatments for AF based on ion channel blockade have demonstrated limited efficacy, underlining the relevance of the development of a prophylaxis and/or novel treatment for this disorder. In the light of the current advances, the future of antioxidant vitamin based POAF preventive therapy looks promising. The studies made in this field, that gathers in-vitro, animal and clinical trials, all point to potential benefits of the antioxidant vitamins to at least prevent or likely treat oxidative stress related disorders. As an amelioration of ischemia/reperfusion tissue injury could be expected to contribute to the success of organ transplantation, future research should aim to find the experimental and clinical support to this view, based on, an under explored measure to optimize the quality of a living organ allograft.

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[20] Polizio AH, Balestrasse KB, Yannarelli GG, et al. Angiotensin II regulates cardiac hypertrophy via oxidative stress but not antioxidant enzyme activities in experimental renovascular hypertension. Hypertens. Res. 2008;31:325-334. [21] Everett TH., & Olgin JE. Atrial fibrosis and the mechanisms of atrial fibrillation. Heart Rhyth. 2007;4:24−27. [22] Olshansky B. Interrelationships between the autonomic nervous system and atrial fibrillation. Prog. Cardiovasc. Dis. 2005;48:57–78. [23] Dilaveris P, Giannopoulos G, Synetos A, Stefanadis C. The role of renin angiotensin system blockade in the treatment of atrial fibrillation. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005;5:387-403. [24] Boos CJ, Anderson RA, Lip GY. Is atrial fibrillation an inflammatory disorder? Eur. Heart J. 2006;27:136-49. [25] Rodrigo R, Cereceda M, Castillo R, et al. Prevention of atrial fibrillation following cardiac surgery: basis for a novel therapeutic strategy based on non-hypoxic myocardial preconditioning. Pharmacol. Ther. 2008;118:104-127. [26] Mihm MJ, Yu F, Carnes CA, et al. Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation. Circulation. 2001;104:174-180.. [27] Ramlawi B, Otu H, Mieno S, et al. Oxidative stress and atrial fibrillation after cardiac surgery: a case-control study. Ann. Thorac. Surg. 2007;84:1166-1172. [28] Kim YM, Guzik TJ, Zhang YH, et al. A myocardial Nox2 containing NAD(P)H oxidase contributes to oxidative stress in human atrial fibrillation. Circ. Res. 2005;97:629-636. [29] Kim YM, Kattach H, Ratnatunga C, Pillai R, Channon KM, Casadei B. Association of atrial nicotinamide adenine dinucleotide phosphate oxidase activity with the development of atrial fibrillation after cardiac surgery. J. Am. Coll. Cardiol. 2008;51:68-74. [30] Bukowska A, Schild L, Keilhoff G, et al. Mitochondrial dysfunction and redox signaling in atrial tachyarrhythmia. Exp. Biol. Med. 2008;233:558-574. [31] Kawakami M, Okabe E. Superoxide anion radical-triggered Ca2+ release from cardiac sarcoplasmic reticulum through ryanodine receptor Ca2+ channel. Mol. Pharmacol. 1998;53:497-503. [32] Peters NS, Coromilas J, Severs NJ, Wit AL. Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation. 1997;95:988-96 [33] Severs NJ, Bruce AF, Dupont E, Rothery S. Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc. Res. 2008;80:9-19. [34] Duffy HS, Wit AL. Is there a role for remodeled connexins in AF? No simple answers. J. Mol. Cell Cardiol. 2008;44:4-13 [35] Ramachandran S, Xie LH, John SA, Subramaniam S, Lal R. A novel role for connexin hemichannel in oxidative stress and smoking-induced cell injury. PLoS ONE. 2007;2:e712 [36] Mejsnar JA, Sopko B, Gregor M. Myofibrillar creatine kinase activity inferred from a 3D model. Physiol. Res. 2002;51(1):35-41.

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In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter V

Acute Renal Failure Joaquín Toro,1 Víctor Molina2 and Ramón Rodrigo3 1

Faculty of Medicine, University of Chile Faculty of Medicine, University of Chile 3 Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile Supported by FONDECYT, grant 1070948 2

Abstract Acute renal failure (ARF) is a condition characterized by a rapid decrease in renal function, leading to an imbalance in water and solutes metabolism. It constitutes a major cause of morbidity and mortality in hospitalized patients worldwide, mainly in elderly population. Despite the medical advances, over the past fifty years the mortality of ARF has not diminished. This is often attributed to increased risk factors prevalence, mainly those derived from changes in our lifestyle. However, it is also possible that the therapeutic methods used until these days are not aiming on the right direction, probably due to lack of knowledge about some of the mechanisms leading to the development and progression of ARF. Over the last decades a large body of evidence has emerged supporting a role of oxidative stress in the pathogenesis of a variety of diseases, including ARF. Indeed, both reactive oxygen and nitrogen species are thought to enhance tubular damage caused from either renal ischemia or direct toxic injury. Nevertheless, the role of oxidative stress in ARF pathogenesis has not been fully established and some evidence is even contradictory. A better understanding regarding the real contribution of oxidative stress to ARF development and progression is required for the design of potentially preventive interventions, such as antioxidant supplementation. Indeed, clinical trials on this matter have been carried out with promising results. This chapter presents an update of the current evidence supporting a role of oxidative stress in ARF pathophysiology, and the potential role of antioxidants in the prevention and treatment of this disease.

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1. Introduction Acute renal failure (ARF) is a condition characterized by a fast declination, from hours to days, of renal function. Consequently, the plasma concentration of nitrogenated compounds is increased (azotemia), hydroelectrolitical and acid-base disorders develop, and extracellular volume (ECV) alterations arise [1, 2]. Approximately 1% of hospitalized patients have ARF at the time of admission, and its estimated incidence during hospitalization is 2-5% [3]. Acute renal failure is said to occur in anywhere from 1% to 25% of critically ill patients [4, 5]. In intensive care settings, the mortality rate of ARF is 70-80%. Current prevention strategies are inadequate and available treatment options besides renal replacement therapy are nonexistent [6]. Despite the medical advances, the mortality of ARF has not diminished in the last forty or fifty years, remaining in about 40-50%. Likely, this is related to an increasing association of this condition with aggravating factors, including increased age, presence of comorbidities, association with multiple renal injuries, inflammatory systemic response syndrome and multiorganic dysfunction syndrome. The lack of reduction in mortality rates might be due to the fact that the underlying mechanisms of ARF, and its final damaging pathway, have not yet been fully elucidated. Indeed, critically ill patients who develop ARF experience a high mortality rate that is not entirely explained by sepsis, advanced age, or underlying morbid conditions [7, 8]. It is well known that elderly patients have increased ARF death risk in comparison to young patients [9]. Moreover, it has been demonstrated that critically ill patients with ARF present an excess of plasma protein oxidation [10]. Since oxidative stress is strongly related to ageing, it could be expected that excessive production of reactive oxygen species (ROS) or impairment in the endogenous ROS scavenging system could play a key role in ARF pathophysiology in these patients. Ischemia and nephrotoxic damage arise as two important causes of ARF, both resulting in oxidative stress. The aim of this chapter is to present an update of the role of oxidative stress in ARF pathophysiology. Also, the mechanisms by which antioxidants supplementation could modify the clinical outcome of these patients are explained.

2. Pathophysiology of Acute Renal Failure Before analyzing the involvement of ROS in ARF pathogenesis, a brief review of basic renal physiology is presented. The kidney is an organ that performs two major functions that are essential for survival: 1. It participates in the maintenance of a relatively constant extracellular environment that is necessary for normal cell functioning. This is achieved through the excretion of waste products of metabolism, such as urea, creatinine and uric acid, and of water and electrolytes, derived mainly from dietary intake. The adequate balance is maintained by keeping the rate of excretion equal to the sum of net intake plus endogenous production, if this occurs.

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2. It produces hormones, enzymes and factors that participate in the regulation of systemic and renal hemodynamics, such as renin, angiotensin II (Ang-II), and prostaglandins. Erythropoietin and calcitriol are of major relevance as renal secretion products. Erythropoietin is related with red blood cell production, and calcitriol is an essential hormone for calcium and phosphate homeostasis. The kidney also performs a number of miscellaneous functions such as catabolism of peptide hormones and synthesis of glucose under fasting conditions, which is known as gluconeogenesis. The morphophysiological unit of the kidney is the nephron. The number of nephrons is estimated in a million per each healthy kidney. For better understanding, the nephron can be divided in two main parts: •

•

Glomerulus: It comprises two zones: the vascular pole and the urinary pole. At the vascular pole, the afferent arteriole (AA) forms the capillary tuft, after which the efferent arteriole (EA) is formed and leaves the glomerulus. The luminal surface of the capillaries is formed by a fenestrated endothelium. The continuous glomerular basement membrane anchors the endothelium to the visceral layer of Bowman’s capsule. This layer is formed by specialized epithelial cells called podocytes, which, along with their numerous extensions (the foot processes) cover the capillaries. Tubules: The urinary space is continuous with the lumen of the proximal tubule. The tubular system is responsible for the reabsorption and secretion processes.

Urine formation begins with filtration of a protein-free plasma, or ultrafiltrate, into the urinary space. The movement of water and associated dissolved small molecules (crystalloids) is determined by hydrostatic and oncotic pressures. Glomerular capillaries are about a hundred times more permeable to water and crystalloids than muscle capillaries. This filtration raises the plasma oncotic pressure as fluid moves along the capillaries, due to net loss of water into Bowman's space. The filtrate is modified as it passes through the nephron by tubular reabsorption and/or tubular secretion. Normal glomerular filtration rate (GFR) is approximately 180 L per day, or 125 mL per minute. Of this enormous amount, only 1-2 L per day are excreted as urine, implying that 99% of the filtered volume is reabsorbed. In pathophysiological terms, ARF is defined as an abrupt decrease in GFR. Impairment of renal function leads to a rise in serum nitrogenated compounds (azotemia), such as creatinine and urea, being the latter frequently measured as blood urea nitrogen (BUN). However, immediately after a kidney injury, BUN or creatinine levels may be normal and the only sign of renal function impairment may be decreased urine output. Moreover, several conditions might alter these parameters, including medications, protein loading or gastrointestinal bleeding. Therefore, creatinine and BUN levels must be interpreted in the context of each patient in order to determine whether or not an alteration of renal function is present. Retention of creatinine and urea is accompanied by the accumulation of a variety of substances generically named as “uremic toxins”. The effects of these toxic substances

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account for many of the symptoms and signs associated with end-stage renal disease. Examples of these uremic manifestations include pericarditis, altered mental status and peripheral neuropathy, among others. Inadequate potassium and sodium excretion are also commonly seen, leading to hyperkalemia and edema, respectively. At this time, the GFR is likely to be found between 5 and 10 mL/min. For reasons that are not well understood, the intrarenal adaptations that allow the maintenance of fluid and electrolyte homeostasis are more likely to occur in chronic renal disease than in ARF. At the same reduction of GFR, patients with ARF are more likely to develop edema, hyponatremia, and hyperkalemia. The terms ARF and acute tubular necrosis (ATN) are often mistakenly exchanged. Acute tubular necrosis is a form of ARF that is caused by an ischemic or toxic injury to the tubular epithelial cells [11]. Acute renal failure may be caused by several etiologies, which can be classified in three large groups:

Pre-Renal Causes Pre-renal causes include a variety of clinical settings that associate with a decreased renal perfusion, as occurs in a diminution of effective arterial volume (EAV) with structurally intact nephrons, giving rise to an adaptive kidney response.

Renal Causes Renal causes are related to cytotoxic, ischemic, or inflammatory insults to the kidney, leading to structural and functional damage. Structural injury to the kidney is the characteristic of intrinsic ARF. The most common form is ATN, either ischemic or cytotoxic.

Postrenal Causes Postrenal causes include all the conditions in which an obstruction to the passage of urine occurs anywhere along the urinary tract, between the renal pelvis and the urethra. Despite the fact that many pathophysiological features are shared among these different categories, they differ in several topics, such as clinical presentation, functional integrity of the tubule, response to therapy and specific diagnosis tests. Then, this classification is useful when establishing a differential diagnosis. The pathophysiological events leading to the death of tubular cells are complex and incompletely understood. Nevertheless, the central hallmarks of either ischemic or toxic ARF are injury, apoptosis and necrosis of tubular cells. As follows, we will discuss the major structural and biochemical features thought to be important for ATN and its consequences.

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2.1. Renal ischemia The kidney is an organ highly responsive to changes in EAV. As a consequence of reduced renal plasma flow (RPF), renal ischemia may give rise to metabolic changes causing a deep impairment in the processes responsible for tubular transport. In addition, structural effects can alter the viability of epithelial tubular cells. Renal ischemia is a disturbance that has generated conflicting experimental data and different pathophysiological explanations. This is reflected in the variety of names given to this condition, including traumatic nephrosis, lower nephron nephrosis, vasomotor nephropathy, post-ischemic ARF and ischemic nephropathy, among others. We will define renal ischemia as the deficiency of blood in one or both kidneys. The impairment of renal perfusion might be due to several causes, mainly arising from functional constriction or genuine obstruction of a renal artery. Other systemic situations leading to renal ischemia include volume loss from internal or external hemorrhage, heart failure, hepatorenal syndrome and shock. The diminution of RPF leads to decreased GFR, being the latter a less marked change. This effect results from a prevailing vasoconstriction of the EA mediated by Ang-II that contributes to the maintenance of the hydrostatic pressure within the glomerular capillaries. Consequently, a rise of the filtration fraction is developed, producing a relative diminution of hydrostatic pressure and a rise in the oncotic pressure at the level of the peritubular capillaries. All of these changes create a favorable condition for sodium reabsorption at the proximal tubule. The diminution in GFR, together with the increased sodium reabsorption, contributes to the elevation of plasma urea and creatinine concentration, producing renal azotemia. In physiological conditions, the kidney has a disproportionately high blood flow in relation to its oxygen consumption [12]. Blood samples from the renal vein have an oxygen tension considerably higher than the mixed venous blood draining other organs. The high renal blood flow is commonly seen as designed to maximize flow-dependent clearance of wastes [13]. Nevertheless, the kidney has also an important functional oxygen reserve. This reserve should protect it from potential ischemic challenges, making it less likely to be damaged by decreased RPF. However, what really occurs is exactly the opposite: the kidney is an organ remarkably susceptible to hypoperfusion, as mentioned before. ¿How is this possible? Initially, it was suggested that a non homogeneous distribution of blood flow exists inside the kidney. Furthermore, several studies have demonstrated that oxygen delivery to the kidney is complex, heterogeneous, and gradient-limited, suggesting the possibility of selective regional hypoxia as a potential major source for localized injury during renal hypoperfusion. Then, although the overall balance of oxygen consumption is relevant, a special attention must be paid to the segment of the nephron that has more chance of being harmed. In general terms, the medullar part of the kidney is more likely to be injured by hypoxia. The most severe damage takes place in the straight part of the proximal tubule, known as the S3 segment. The model that has been proposed for explaining the pathophysiology of renal ischemia is focused in alterations occurring in the tubular epithelium. Early morphologic changes observed after ischemia include the formation of blebs in the apical membranes of proximal

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tubule cells, with loss of the brush border [14, 15]. Afterwards, proximal tubule cells lose their polarity, and the integrity of their tight junctions is disrupted [16], a process that is thought to arise from alterations in the actin and microtubule cytoskeletal organization [17, 18]. As a consequence, some cellular-membrane proteins are transferred to unusual sites. For instance, the Na+/K+ - ATPase redistributes from the basolateral to the apical membrane [19] thus reducing or even more, reversing the unidirectional sodium transport from tubular lumen to peritubular interstitial space. The increased sodium delivery to the distal tubule triggers the tubule-glomerular feedback, which leads to a vasoconstriction of the AA, with the consequent decrease of GFR. This is the most relevant mechanism of the maintenance phase of ARF. When ischemic damage occurs, integrins, a group of proteins involved in intercellular adhesion, move to the apical surface of the tubular epithelium [20], and facilitate its adhesion with cells that have been shed due to apoptosis or necrosis, thereby forming conglomerates that cause obstruction in the tubular lumen [21]. Then, the desquamation of tubular epithelium leads to a raise in intratubular hydrostatic pressure. In addition, backleak of filtrate occurs as a consequence of structural alterations affecting tubular integrity, further contributing to the diminution of urine output currently present in this setting. Several changes occur due to the lack of ATP caused by oxygen deprivation, particularly in the most metabolically active tubular cells. After the occurrence of hypoxia, but before cell membrane damage, the elevation of intracellular sodium concentration contributes to the development of an increased intracellular calcium concentration [22]. In turn, intracellular calcium activates phospholipase A2 that hydrolyze phospholipids of the plasma membrane, releasing fatty acids and lisophospholipids. It was reported that peroxidation of membrane lipids due to ischemia-reperfusion enhances the susceptibility of membranes to phospholipase A2 (PLA2) [24]. Additionally, arachidonic acid, a product of PLA2, is converted into eicosanoids that produce vasoconstriction and are chemotactic for neutrophils [25]. Calcium can also contribute to epithelial cell toxicity through its ability to activate proteases, break down the cytoskeleton, and interfere with mitochondrial energy metabolism. However, there is still controversy regarding the in vivo intracellular calcium concentration required to cause ischemic tubular cell injury, and if it is possible to reach this concentration in tubular cells [26]. In ischemic ARF there is also a neutrophil infiltration in the kidney. The migration of leucocytes to neighbor tissues is possible through the binding of neuthrophil integrins to adhesion molecules present in the vascular endothelium. This migration to the interstitial space leads to cell damage by increased ROS production and the activation of enzymes such as collagenases, elastases and myeloperoxidases, thereby promoting the migration of further inflammatory cells. Although leucocytes appear to have an important role in AFR pathogenesis, neutropenic patients can also develop severe forms of ARF. Then, it seems that leucocytes are not essential for the development of acute tubular disease. However, their role in ARF has yet to be fully established.

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2.2. Nephrotoxic Damage Nephrotoxic damage exerted by toxins accounts for the second more important mechanism of ARF development. It is important to notice that the mechanisms whereby the toxins cause tubular necrosis share many pathophysiological features with ischemic ARF [27]. Moreover, ischemia and toxins often combine to cause ARF in severely ill patients with conditions such as sepsis, hematological disorders, cancer or acquired immunodeficiency syndrome (AIDS) [28, 29]. The mechanisms by which drugs can cause ARF are detailed next. 2.2.1. Direct Tubular Cell Damage Aminoglycoside antibiotics and radiocontrast agents are the most common toxins that cause ARF and both induce damage frequently in proximal tubule. Furthermore, vancomycin, cisplatin, immunoglobulin and mannitol may also generate proximal damage leading to impaired tubular function. Typically, tubular cells lose polarity, develop vacuoles and eventually separate from the basement membrane. Marked disturbance of electrolyte homeostasis may also occur due to effects on water reabsorption in the distal tubule [30]. Acute anuria has been reported in critically ill patients treated with high dose immunoglobulin for Guillain-Barré syndrome, probably due to acute renal dysfunction caused by proximal tubular cell damage, a mechanism known to occur also in mannitol nephrotoxic damage [31]. On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs), angiotensin converting enzyme (ACE) inhibitors, cyclosporin A, lithium, and cyclophosphamide induce direct damage essentially in the distal tubule. Consequently, a disturbance of sodium, potassium, hydrogen ion and water balances is produced. Non steroidal anti-inflammatory drugs, ACE inhibitors and cyclosporin A, all alter potassium balance, resulting in hyperkalemia. Chronic administration of lithium may initially result in transitory and then in permanent inability to thrive, causing insipidus nephrogenic diabetes. Acute lithium intoxication causes a similar tubular effect which may be reversible. In contrast, high doses of cyclophosphamide may result in hyponatremia due to an impaired ability to excrete water. In general terms, direct cellular damage is dose-dependent and is enhanced when occurring in hypoxic conditions. For instance, amphotericin B is capable of inducing damage in both proximal and distal tubules. The enhanced membrane permeability produced by amphotericin B triggers an increase in active sodium transport and oxygen demand. In consequence, a more severe damage occurs if a reduced supply of oxygen is associated [32, 33]. Renal damage arising from the nephrotoxic effects of radiocontrast agents is becoming increasingly common. Radiocontrast media is used in relatively high doses for computed tomography scans and some types of vascular surgery. The risk of renal damage is particularly high in patients who already have impaired renal function or those with diabetes mellitus [34]. In patients with both diabetes and impaired renal function, the incidence of further renal failure following use of radiocontrast agents is over 50%. In these cases, both vasoconstriction and direct tubular damage occur. Preventive measures are limited to saline diuresis prior to radiocontrast administration.

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There are a number of other drugs such as tacrolimus, methotrexate, foscarnet, and pentamidine that are known to be potentially nephrotoxic [35]. Additionally, several other substances can cause direct tubular damage including organic solvents, heavy metals (e.g. mercury) and carbon tetrachloride. Finally, direct tubular damage can also be caused by some plant and animal toxins. 2.2.2. Reduction in Renal Perfusion through Alteration of Intrarenal Hemodynamics In volume depleted states some drugs can also induce ARF through alterations in intrarenal hemodynamics. This is the case of ACE inhibitors and angiotensin receptor blockers. It is remarkable to notice that these drugs are otherwise safely tolerated and beneficial in most patients with chronic kidney disease. Angiotensin II acts directly within the glomerular circulation. The use of ACE inhibitors, not only inhibits Ang-II production but also interferes with bradykinin, which has an important role in the circulatory control of the glomerulus. Non-steroidal anti-inflammatory drugs may induce a decrease in GRF through a selective inhibition of cyclo-oxygenase that normally acts as a vasodilator in the AA, thereby inhibiting the compensatory mechanisms that protect the kidney from reduced plasma flow in volume depleted states. Cyclosporine A has a similar effect in the AA. Both of these drugs and their effects are potentiated by hypovolemia, low cardiac output, sepsis, liver disease, and pre-existing renal failure. Arteriolar vasoconstriction leading to ARF may also occur in hypercalcemic states, with the use of radiocontrast agents, amphotericin B, calcineurin inhibitors, norepinephrine, and pressor agents, among others. 2.2.3. Intratubular Obstruction by Precipitation of the Agent, Its Metabolites or by-Products Drugs that may directly or indirectly cause tubular obstruction include acyclovir, sulfonamides, ethylene glycol, chemotherapeutic agents, and methotrexate. Patients with Pneumocystis pneumonia as a result of AIDS and other immunosuppressive disorders are increasingly being treated with high doses of sulphonamides. Such treatment is associated with increased incidence of crystalluria resulting in tubular obstruction and renal dysfunction. Adequate salt and water loading should preserve the tubular filtrate flow, thereby preventing the precipitation of drug and hence renal failure. The most common used anti-viral agent, acyclovir, and the protease inhibitor indinavir, a pillar in AIDS treatment, have both similar toxic actions. Treatment of patients with high dose chemotherapy for hematological malignancies can result in rapid cytolytic effect resulting in a greatly increased uric acid load arriving to the kidney. In such patients, acute crystalluria may develop unless adequate urine flow and sodium diuresis is maintained. Ethylene glycol, a known antifreeze liquid, can be a cause of ARF when it is accidentally taken, such as occurs in children poisoning. Its metabolism results in a large oxalate load which may crystallize in the tubule, thereby causing obstruction.

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2.2.4. Allergic Interstitial Nephritis Acute renal failure due to acute interstitial nephritis is most often caused by an allergic reaction to a drug [36]. In strict sense, every drug might induce interstitial nephritis. Nevertheless, the drugs that are more likely to produce this syndrome are: penicillins, cephalosporins, sulfonamides, rifampicin, ciprofloxacin, vancomycin, NSAIDs, thiazide diuretics, furosemide and allopurinol. Less commonly ranitidine, cimetidine and phenytoin may also cause similar damage. 2.2.5. Heme Pigment-Induced Tubular Toxicity (Rhabdomyolysis) Rhabdomyolysis-induced ARF is a condition that will be further described below in this chapter. It is caused by drugs such as cocaine, ethanol and statins, particularly lovastatin, which may induce ARF. Rhabdomyolysis is more likely to occur when lovastatin is given in combination with cyclosporine [37]. 2.2.6. Hemolytic–Uremic Syndrome Certain drugs can cause hemolytic-uremic syndrome, including cyclosporine, tacrolimus, mitomycin, cocaine, quinine and conjugated estrogens. Non-drug related causes include autoimmune diseases (e.g. lupus, Wegener granulomatosis), infiltrative diseases (e.g. sarcoidosis), hematologic diseases (e.g. myeloma through light-chain proteins) and infectious agents (e.g. legionnaire’s disease and Hantavirus infection) [38-40].

2.3. Role of Oxidative Stress in the Mechanism of Renal Damage There is evidence supporting a role of ROS in kidney cellular injury. This includes the demonstration of an accentuation of renal injury by oxidants and by antioxidants deficiency. Accordingly, Himmelfarb et al. [10] measured the concentrations of a group of oxidative stress biomarkers in the setting of ARF. In their retrospective analysis of PICARD study (Program to Improve Care in Acute Renal Disease) samples, they determined the plasma protein thiol content, which is a marker of total antioxidant capacity, and the plasma protein carbonyl content, which is an index of oxidative injury, in critically ill patients with and without associated ARF, patients with end stage renal disease and healthy controls. Critically ill patients with associated ARF displayed a significant decrease of thiol content and an increase of carbonyl content, in relation to all the other groups. In the kidney, as well as other organs, ROS can react with proteins, carbohydrates, nucleic acids, and cell membrane lipids. This results in organic radical formation, enzyme inactivation, glutathione oxidation, lipid peroxidation, and renal cell destruction [41]. Therefore, consequences of ROS activity include proteinuria, disturbances in GFR and morphological changes in the glomerulus [42]. Acute renal failure itself is recognized as an additional stimulus for oxidative stress [43, 44]. This is a consequence of the dysregulated inflammatory response in these patients, which basically consists in stimulated phagocytic cells, leading to excess cytokines production. Indeed, these cells are major producers of ROS.

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Furthermore, oxidative stress is considered an important pathogenic mechanism for the development of ischemic and toxic renal tubular injury [45-47]. As follows, the involvement of ROS in the diverse mechanisms leading to renal damage will be analyzed separately.

2.3.1. Ischemia-Reperfusion Increasing evidence has accumulated over the last few years indicating that ROS could play a crucial role in a variety of pathogenic mechanisms, including ischemia-reperfusion injury in several human organs. In ischemic tissue conditions, such as myocardial infarction or prerenal ARF, most of the cell injury is not inflicted during the period of ischemia, but after the blood flow to the damaged tissue is restored. This is called reperfusion injury. Ischemia shifts cellular metabolism from aerobic to anaerobic with rapid depletion of intracellular ATP stores and increased hypoxanthine concentrations [48]. During reperfusion, the oxygen delivery enables the activity of xanthine oxidase (XO), an enzyme that catalyzes the conversion of hypoxanthine to xanthine and uric acid, resulting in an intensification of superoxide anion (O2•–) and hydrogen peroxide (H2O2) generation [49]. Indeed, the production of these two highly reactive species starts only when oxygen is widely available [50]. The respiratory burst also activates polymorphonuclear leukocytes and monocytes that penetrate the glomerulus and interstitium to become another source of large quantities of ROS [51]. Another pathway for the production of ROS during reperfusion following ischemia is cyclooxygenase and lipoxygenase activation [52]. This excessive production of ROS causes oxidative stress that results in several changes, including impairment of mitochondrial oxidative phosphorylation, ATP depletion, increase in intracellular calcium, and activation of proteases and phosphatases. These changes lead to the breakdown of membrane phospholipids and cellular cytoskeleton, resulting in loss of cellular integrity [26, 53-56]. Although the contribution of early generation of reactive nitrogen species (RNS) to the development of renal failure has yet to be fully established, it is tempting to speculate that the generation of RNS, rather than hydroxyl radical, is more important for the injury associated with ischemia-reperfusion damage [57]. In normal kidney functioning, endothelium-dependent vasodilators, such as acetylcholine and calcium ionophore A23187, act by stimulating endothelial nitric oxide synthase (eNOS) activity, thereby increasing endothelium-derived NO production. In contrast, other vasodilators such as nitroprusside and nitroglycerin induce vasodilation by directly releasing NO in vascular smooth muscle cells, this way acting through an endothelium-independent mechanism. Nitric oxide produced by eNOS, as well as released by these NO donor agents, induces vasodilation by stimulating the production of cyclic guanosine monophosphate (cGMP) in vascular smooth muscle cells. Other substances, like atrial natriuretic peptide (ANP), are also endothelium-independent vasodilators but do not act through a mechanism involving NO. ANP directly stimulates an isoform of guanylyl cyclase in vascular smooth cells, inducing vasodilation [58]. Over the last decade, several studies have agreed that eNOS activity is impaired following ischemia-reperfusion cycle (for more details see chapter 2). Impaired production of NO contributes to the vasoconstriction associated with established ARF. There is evidence showing that, in isolated erythrocyte-perfused kidney, ischemia-

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reperfusion injury is associated with intrarenal vasoconstriction that is reverted by endothelium-independent vasodilators, but not by endothelium-dependent vasodilators. This data suggests that endothelium-derived NO production is impaired following ischemic injury and that the inhibition of eNOS activity can contribute to the vasoconstriction associated with ARF [59]. Although impaired NO production can contribute to renal damage through vasoconstriction, NO itself can also be involved in an injury mechanism related with ROS. During ischemia-reperfusion NO can react with ROS, this way decreasing its bioavailability and leading to vasoconstriction. However, this reaction also leads to the production of peroxynitrite, a highly reactive oxidant molecule. Evidence supporting this mechanism shows that in a model of isolated proximal tubules the injury due to hypoxia-reoxygenation can be prevented through the inhibition of nitric oxide synthase activity and by the addition of hemoglobin, an NO scavenger. Moreover, L-arginine, the nitric oxide synthase substrate, and nitroprusside, a NO donor, can enhance tubular injury under these conditions [60]. In physiological conditions, sulfhydryl groups react with NO in the presence of oxygen to produce S-nitrosothiols, which are stored in cells as S-nitrosoglutathione [61]. It is thought that renal tissue nitrosothiols release NO when renal blood flow is altered, oxygen tension falls to zero, and NO synthesis is ceased. In this situation, S-nitrosothiols decompose slowly to release NO [62]. If released NO is not inactivated by oxygen or oxyhemoglobin, its concentration should increase progressively until reaching a maximum [63]. Some in vitro studies have shown that there exists a synergistic interaction between PLA2 and ROS in ischemia-reperfusion injury of the kidney. Membranes exposed to ROS are peroxidized and become more susceptible to PLA2 action. This synergy occurs also in mitochondria, where PLA2 acts in concert with ROS to uncouple oxidative phosphorylation [64]. Indeed, it has been reported that renal ischemia-reperfusion results in increased PLA2 activity of the cytosolic, mitochondrial, and microsomal subcellular fractions of the kidney [65, 66]. This has led to the observation that mitochondrial PLA2 activation could play a major role in post-ischemic cellular injury. Furthermore, it was demonstrated that hyperbaric oxygen does not induce a significant change in PLA2 activity in the non ischemic kidney, indicating that this type of oxidative stress alone does not cause PLA2 activation in the mitochondria. However, when hyperbaric oxygen was combined with ischemia-reperfusion, mitochondrial PLA2 activity was markedly enhanced. This suggests that the activation of PLA2 caused by ischemia-reperfusion is enhanced by ROS. Moreover, a study demonstrated that the exposure to high oxygen concentration resulted in a significant decrease in superoxide dismutase (SOD) activity in the post-ischemic rat kidney, probably due to consumption by excessive amounts of ROS [67]. Finally, surgical interventions, such as renal transplantation, can also associate with ischemia-reperfusion injury. This may constitute an important factor predisposing to organ rejection [68]. 2.3.2. Rhabdomyolysis Myoglobinuria plays a key role in the pathophysiology of acute renal failure in clinical settings that are characterized by muscle tissue injury [50]. The term rhabdomyolysis refers to disintegration of striated muscle, which results in the release of muscular cell constituents into the extracellular fluid and the circulation. One of the key compounds released is myoglobin,

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an 18,800-Dalton oxygen carrier. It resembles hemoglobin, but contains only one heme moiety. Apart from myoglobin, during rhabdomyolysis potentially toxic myocyte contents are released into the systemic circulation. The renal consequences of this disturbance have been attributed to both intense vasoconstriction and renal tubular necrosis. Normally, myoglobin is loosely bound to plasma globulins and only small amounts reach the urine. However, when massive amounts of myoglobin are released, the binding capacity of the plasma globulins is exceeded. Myoglobin is then filtered by the glomerulus and reaches the tubules, where it may cause obstruction and renal dysfunction [69]. The intratubular degradation of myoglobin results in a massive generation of ROS that overwhelms the scavenging capacity of the antioxidant system, thereby generating renal damage. In fact, it has been proved that myoglobin can induce proximal tubular cell death through the generation of H2O2 [70]. 2.3.3. Dialysis Dialysis procedure is commonly related with chronic renal failure. Nevertheless, dialysis also represents a therapeutic option for ARF when drugs and other treatments have failed. Indeed, it constitutes a common therapeutic method in hospitalized patients with intrinsic ARF. The procedure is repeated as many times as necessary until the patient recovers its renal function. Oxidative stress contributes to morbidity in hemodialyzed patients. In order of importance, three possible sources of ROS can be present in hemodialysis: the uremic state, the dialyzer membrane, and bacterial contaminants from the dialysate [71]. In general terms, favorable conditions for oxidative stress development are generally present in uremic patients on maintenance hemodialysis. In this setting, increased generation of oxidants is associated with chronic antioxidant deficiency [67, 72]. The generation of ROS during hemodialysis sessions can be measured by basal whole blood chemiluminescence (CL) [73, 74]. It has been demonstrated that the production of ROS by phagocytic cells is strictly dependent on the cellulosic nature of the dialysis membrane [73], and is closely related to the amount of the C5a and C3a complement fractions [75]. It has been reported that the increased intracellular ROS production in both neutrophils and monocytes from dialysis patients is associated with increased expression of adhesion molecules, which are key mediators for renal damage, as mentioned before in this chapter [76]. The excessive production of ROS also promotes alterations in the endothelium, which is known to be the first step toward atherosclerosis (see atherosclerosis chapter). Then, although the generation of ROS due to hemodialysis might be intermittent, the consequences of their action are beyond to be transitory. Indeed, it has been found that after hemodialysis there is a decrease of plasma ROS scavenging capacity [77]. This effect is thought to be related to the loss of antioxidants due to the dialysis process. In consequence, antioxidant supplementation could be an important therapeutic approach in the prevention of dialysis induced oxidative stress.

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2.3.4. Diabetic Nephropathy Diabetic nephropathy is a chronic renal disease model, and it represents the most common form chronic renal failure. Thus, it is pertinent to make a brief mention of ROS involvement in its pathophysiology even though it does not constitute an ARF cause. There is increasing amount of evidence supporting a key role of oxidative stress in the development and progression of diabetic nephropathy. High glucose induces intracellular ROS production, either directly via glucose metabolism and auto-oxidation, or indirectly through the formation of advanced glycation end products (AGE) and their receptor binding. Reactive oxygen species mimic the stimulatory effects of elevated glycemia and upregulate TGF-β, PAI-1, and other extracellular matrix proteins in glomerular mesangial cells, thus leading to mesangial expansion and subsequent renal damage [78]. Indeed, it has been suggested that the trigger for hyperglycemia induced damage in the diabetic kidney is the excessive generation of mitochondrial O2•–. Superoxide leads to the activation of four major biochemical pathways, including increased AGE formation, activation of protein kinase C isoforms, and increased flux through the polyol and hexosamine pathways. In addition, each of these pathways can contribute to ROS generation [79] Then, maintenance of oxidative phosphorylation and normalization of mitochondrial function could be key strategies to reduce the progression of diabetic nephropathy. Additionally, further investigation regarding other cellular pathways, such as NADPH oxidase and uncoupling of eNOS, is required to assess their relevance in diabetic nephropathy and in other models of progressive human renal disease [80]. 2.3.5. Nephrotoxic Damage Aminoglycoside antibiotics are probably the most recognized potentially nephrotoxic drugs. Unfortunately, their use is limited not only for causing nephrotoxicity (which occurs in 10–15% of cases), but also for irreversible ototoxicity (in approximately 3–25% of patients) [81]. While gentamicin and other aminoglycosides have been studied extensively, the biochemical and cellular basis of their nephrotoxicity are not completely understood. However, it is evident that gentamicin leads to the disruption of the proximal convoluted tubule and interferes with critical cellular processes through several mechanisms, including oxidant injury. Gentamicin enhances the generation of ROS by altering mitochondrial respiration, leading to the generation of H2O2. Also, it induces the release of iron from renal cortical mitochondria, causing lipid peroxidation in vitro, with iron serving as a potent catalyst for free radical formation [82].

3. Effects of Antioxidants in Acute Renal Failure Several investigations have suggested a role of endogenous and/or exogenous antioxidants in renal protection. However, evidence is not concluding and thereby the role of antioxidants in acute renal failure still has to be established. It is remarkable to notice that the beneficial role of antioxidants is out of discussion in chronic renal disease.

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The major endogenous mechanism for the removal of ROS is the antioxidant enzyme system. This system includes two superoxide dismutases that convert O2•– to H2O2 (Cu/ZnSOD and Mn-SOD), and two more enzymes, catalase (CAT) and glutathione peroxidase (GSH-Px), that degrade H2O2 to H2O (for more details see chapter 1). For example, it has been observed that SOD inhibits ROS generation, decreases lipid peroxidation in cortical mitochondria and protects the kidney from injury after blood reflow. In this study CAT activity did not protect against ischemia-reperfusion injury [83]. Nevertheless, a few years later the same authors demonstrated that the inhibition of CAT before ischemia leads to an exacerbation of the ischemic injury [84]. Accordingly, Baker et al. showed that kidney tissue taken from animals after ischemia alone was extensively damaged compared with tissue from SOD-treated animals [53]. Also, it has been confirmed that an elevated intracellular GSH concentration protects rat renal proximal tubules against in vitro simulated reperfusion injury [85]. In contrast, it has been reported a non significant fall in the activity of SOD with no changes in the activity of CAT in erythrocytes of renal transplant patients [68]. Disparities among results can be attributed to the diverse time gaps used in ischemiareperfusion experimental models. Indeed, it has been found that 30 minutes of ischemia followed by reperfusion has little effect on enzyme activity, whereas longer duration of ischemia (60 or 90 minutes) results in significant loss of the expression and activity of catalase, GSH-Px and Cu/Zn-SOD. In the same experimental model, Mn-SOD expression and activity experienced an induction [86]. Moreover, it has been suggested that both instability of mRNA for catalase, GSH-Px and Cu/Zn-SOD, and higher transcriptional activity of Mn-SOD genes are associated with the modulation of antioxidant response to ischemia-reperfusion injury in kidney [87] During reperfusion following ischemia, superoxide anion is thought to be mainly produced by XO. This is supported by several studies showing an increased activity of XO during reperfusion and an attenuation of ROS generation under these conditions with the use of allopurinol, a XO inhibitor [83, 88]. Moreover, it has been suggested that allopurinol could play a role in the prevention of kidney damage during ischemia-reperfusion cycle. The protective effect of the modulation of antioxidant enzymes against ischemiareperfusion induced oxidative stress provide further evidence of the impact of these enzymes on the degree of tissue damage [89-92]. Conflicting data has been reported regarding the levels of antioxidants in dialysis patients. Endogenous antioxidant scavengers may be low in dialysis patients due to diminished oral intake, dietary restrictions, dialytic clearance, or as a result of increased degradation. For instance, vitamin C deficiency may be secondary to dietary restriction of fresh fruits and vegetables to avoid hyperkalemia, but also to loss of the vitamin during dialysis. Plasma vitamin E concentrations are typically normal, whereas erythrocyte and mononuclear cell concentrations appear to be decreased [93]. Vitamin E appears to be important in the protection against oxidation of low-density lipoproteins (LDLs) and biological membranes. It has been reported that both oral and parenteral administration of vitamin E improves renal anemia and erythropoietin requirements in dialysis patients [94]. More recent reports indicate that vitamin E given orally also attenuates oxidative stress induced by intravenous iron administration and

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significantly decreases the oxidative susceptibility of LDL [95, 96]. However, in a randomized double-blind placebo-controlled trial, intravenous supplementation with vitamin E in elective cardiac surgery failed to show a decrease in biochemical markers of oxidative stress and in the rate of ARF [97]. It has been observed that postdialysis intravenous administration of vitamin C, compared to ferric saccharate, significantly increases the hematocrit and diminishes erythropoietin requirements after 8 weeks of therapy [96]. Vitamin C acts by promoting iron release from storage sites, by elevating its delivery to hematopoietic tissues, and by increasing iron utilization in erythroid progenitor cells. While these studies showed beneficial effects of antioxidant supplementation strategies, it should be mentioned that both vitamin E and vitamin C can be pro-oxidant under certain adverse circumstances [99,100] Supplementation with N-acetylcysteine (NAC), a glutathione precursor, is another pharmacological antioxidant approach. N-acetylcysteine has been demonstrated to exert beneficial effects in the prevention of oxidant mediated renal injury [101]. Accordingly, Feldman et al. [102] conducted a prospective randomized controlled open label trial investigating the role of N-acetylcysteine in the prevention of gentamicin-induced hearing loss in the setting of end stage renal disease. While the exact mechanism of ototoxicity is different from that of nephrotoxicity, it should be noted that their common pathogenesis includes oxidative stress and ROS. This trial showed that administration of NAC at a dose of 600 mg twice a day was effective in decreasing the rate of ototoxicity at both 1 and 6 weeks. Similarly, Mazzonet et al. demonstrated a protective effect of NAC on gentamicin-induced nephrotoxicity in rats [103]. However, there are no studies demonstrating a nephroprotective effect in humans. Several animal models have attempted to use antioxidant strategies to attenuate cisplatin nephrotoxicity. Ajith et al. [104] conducted a comparative study of the effects of different doses of vitamins C and E on cisplatin-induced nephrotoxicity in mice. High doses of both vitamins were effective in protecting against oxidative renal damage, as measured by increased SOD activity and concentration of reduced glutathione (GSH), with vitamin C outperforming vitamin E. Similarly, Lynch et al. demonstrated in a rodent model that cisplatin nephrotoxicity could be attenuated by the use of allopurinol and ebselen, a selenoorganic glutathione mimic and ROS scavenger drug [105]. In this model the combination of allopurinol and ebselen outperformed the administration of each drug individually, leading to a significant decrease in post-cisplatin serum creatinine and BUN elevations. Moreover, amifostine, an FDA approved agent for the reduction of renal toxicity in patients receiving cisplatin, decreases nephrotoxicity by donating protective thiol groups. Unfortunately, amifostine use is limited by a variety of factors including its cost, side effect profile, and concerns that its use could interfere with the antitumor effects of cisplatin [106]. Many prophylactic and therapeutic experimental animal studies have suggested the possibility of attenuating gentamicin-induced renal failure through antioxidant based therapeutic approaches. Zurovsky and Haber demonstrated that vitamin E and dimethylthiourea, a potent scavenger of hydroxyl radicals, were effective in preserving renal function and arresting progressive renal damage associated with gentamicin administration [107].

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Much of the evidence regarding rhabdomyolysis pathophysiology has been achieved in animal models of myoglobinuric ARF. The most common in vivo model uses intramuscular injection of hypertonic glycerol. In these conditions it has been reported an increased generation of H2O2, lipid peroxidation and depletion of GSH stores [108]. Red wine polyphenols lead to a decreased vulnerability of the rat kidney to ATN caused by rhabdomyolysis-induced myoglobinuria [109]. Also, resveratrol, a stilbene polyphenol found in grapes and red wine, has been found to reduce the mortality due to renal ischemiareperfusion injury in rats, as well as to improve renal function and decrease histological tissue damage. This effect appears to be mediated by an increased production of NO in resveratrol treated rats [110]. In addition, it has been demonstrated that the rat kidney responds to glycerol-induced rhabdomyolysis with an induction of heme oxygenase as well as the synthesis of ferritin [111]. This constitutes a protective antioxidant response and suggests a therapeutic strategy for populations at a high risk for rhabdomyolysis. Unfortunately, evidence regarding a potential protective effect of antioxidant-based therapeutic approaches in rhabdomyolysis arises mainly from animal models and has not led yet to clinical studies. Indeed, the relevance of ROS in rhabdomyolysis has to be further investigated. Recently, it has been reported a potentially protective effect of aminoguanidine, an inducible nitric oxide synthase inhibitor with antioxidant properties, in ischemia-reperfusion renal injury. Aminoguanidine has shown to reduce serum urea, creatinine levels and improve histopathological lesions when administrated after an ischemia-reperfusion injury to the rat kidney, but not before [112].

4. Conclusion and Perspectives Acute renal failure constitutes a major cause of morbidity and mortality, especially in hospitalized and elderly patients. Oxidative stress is a metabolic derangement that plays a key role in the development and progression of renal damage. The pathophysiology of ARF is a wide and highly complex subject, especially due to the multiple mechanisms that can be involved in its development. Reactive oxygen species appear to play a key role as mediators in intracellular signaling, leading to deleterious effects such as apoptosis and necrosis. Consistently, there is an increasing body of evidence supporting a potential role of antioxidant-based therapy in the treatment and prevention of ARF. However, most of the available data has been achieved in animal and in vitro models. There are only a few studies in humans and data are not sufficient to account for the efficacy of antioxidants as preventive and therapeutic tools. In consequence, further investigation is required to fully establish the role of oxidative stress in the pathophysiology of ARF and the potentially protective and therapeutic effects of antioxidant-based therapies in this setting.

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Figure 5-1. Pathophysiology of acute renal failure (ARF). Any of the three classical causes of ARF leads to two common mechanisms, ischemic damage and nephrotoxic damage. These mechanisms can activate molecular mediators or directly damage the tubule. Both of these conditions result in a rise in reactive oxygen species (ROS) concentrations. Therefore, molecular mediators, ROS and direct tubular injury lead to ARF development and progression.

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[42] Heinzelmann M, Mercer-Jones MA, Passmore JC. Neutrophils and renal failure. Am. J. Kidney Dis. 1999;34:384-399. [43] Himmelfarb J, Ikizler TA, Stenvinkel P, Hakim RM. The elephant in uremia: Reflections on oxidant stress as a unifying concept of cardiovascular disease in uremia. Kidney Int. 2002;62:1524–1538. [44] Descamps-Latscha B, Drueke T, Witko-Sarsat V. Dialysis-induced oxidative stress: Biological aspects, clinical consequences, and therapy. Semin. Dial. 2001;14:193–199. [45] Zager RA, Burkhart K. Myoglobin in proximal human kidney cells: roles of Fe, Ca2+, H2O2, a mitochondrial electron transport. Kidney Int. 1997;51:728–738. [46] Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Drug Metab. Rev. 1999;31:971–997. [47] Noiri E, Nakao A, Uchidna K, Tsukahara H, Ohno M, Fujita T, Brodsky S, Goligorsky MS. Oxidative and nitrosative stress in acute renal ischemia. Am. J. Physiol. 2001;281:F948–F957. [48] Andreoli SP. Reactive oxygen molecules, oxidant injury and renal disease. Pediatr. Nephrol. 1991;5:733-742. [49] Greene EL, Paller MS. Xanthine oxidase produces O2- in posthypoxic injury of renal epithelial cells. Am. J. Physiol. 1992;263:F251-F255. [50] Vanholder R, Sever MS, Erek E, Lamiere N. Rhabdomyolysis. J. Am. Soc. Nephrol. 2000;11:1553–1561. [51] Andreoli SP, McAteer JA, Mallett C. Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro. Kidney Int. 1990;38:785-794. [52] Okusa MD. The inflammatory cascade in acute ischemic renal failure. Nephron. 2002;90:133-138. [53] Baker GL, Corry RJ, Autor AP. Oxygen free radical induced damage in kidneys subjected to warm ischemia and reperfusion. Protective effect of superoxide dismutase. Ann. Surg. 1985;202:628-641. [54] Johnson KJ, Weinberg JM. Postischemic renal injury due to oxygen radicals. Curr. Opin. Nephrol. Hypertens. 1993;2:625–635. [55] McCord JM. Human disease, free radicals, and the oxidant/antioxidant balance. Clin. Biochem. 1993;26:3511–3517. [56] Paller MS. The cell biology of reperfusion injury in the kidney. J. Invest. Med. 1994;42:632–639. [57] Walker LM, York JL, Imam SZ, Ali SF, Muldrew KL, Mayeux PR. Oxidative stress and reactive nitrogen species generation during renal ischemia. Toxicol. Sci. 2001;63:143-148. [58] Kim Y-M, Tseng E, Billiar TR. Role of NO and nitrogen intermediates in regulation of cell functions, in: Nitric Oxide and the Kidney. Edited by Goligorsky M, Gross S. 1997; New York: Chapman and Hall. [59] Lieberthal W, Wolf EF, Rennke HG, Valeri CR, Levinsky NG. Renal ischemia and reperfusion impair endothelium-dependent vascular relaxation. Am. J. Physiol. 1989;256:F894-F900.

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[76] Zwacka RM, Reuter A, Pfaff E, Moll J, Gorgas K, Karasawa M, Weiher H. The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species. EMBO J. 1994;13:5129–5134. [77] Chen TS, Liou SY, Chang YL. Chemiluminescent analysis of plasma antioxidant capacity in uremic patients undergoing hemodialysis. Ren. Fail. 2008;30:843-847. [78] Ha H, Lee HB. Oxidative stress in diabetic nephropathy: basic and clinical information. Curr. Diab. Rep. 2001;1:282-287. [79] Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature. 2000;404:787–790. [80] Forbes JM, Coughlan MT, Cooper ME. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes. 2008;57:1446-1454. [81] Humes HD. Aminoglycoside nephrotoxicity. Kidney Int. 1988;33:900–911. [82] Walker P, Barri Y, Shah S. Oxidant mechanisms in gentamicin nephrotoxicity. Renal Failure. 1999;21:433–442. [83] Paller MS, Hoidal JR, Ferris TF. Oxygen free radicals in ischemic acute renal failure in the rat. J. Clin. Invest. 1984;74:1156-1164. [84] Paller MS. Hydrogen peroxide and ischemic renal injury: effect of catalase inhibition. Free Radic. Biol. Med. 1991;10:29-34. [85] Paller MS, Patten M. Protective effects of glutathione, glycine or alanine in an in vitro model of renal anoxia. J. Am. Soc. Nephrol. 1992;2:1338-1344. [86] Singh I, Gulati S, Orak JK, Singh AK. Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury. Mol. Cell. Biochem. 1993;125: 97–104. [87] Dobashi K, Ghosh B, Orak JK, Singh I, Singh AK. Kidney ischemia-reperfusion: modulation of antioxidant defenses. Mol. Cell Biochem. 2000;205:1-11. [88] Linas SL, Whittenburg D, Repine JE. Role of xanthine oxidase in ischemia/reperfusion injury. Am. J. Physiol. 1990;258:F711-F716. [89] Greenwald RA. Superoxide dismutase and catalase as therapeutic agents for human diseases. Free Rad. Biol. Med. 1990;8:201–209. [90] Reilly PM, Schiller HJ, Buckley JB. Pharmacologic approach to tissue injury mediated by free radicals and other reactive oxygen metabolites. Am. J.Surg. 1991;161:488–503. [91] Shanley PF, White CW, Avraham KB, Groner Y, Burke TJ. Use of transgenic animals to study disease models: Hyperoxic lung injury and ischemic acute renal failure in ‘high SOD’ mice. Renal Fail. 1992;14:391–394. [92] Yoshioka T, Homma T, Meyrick B, Takeda M, Moore-Jarrett T, Kon V, Ichikanea I. Oxidants induce transcriptional activation of manganese superoxide dismutase in glomerular cells. Kidney Int. 1994;46:405–413. [93] Cohen JD, Viljoen M, Clifford D, de Oliveria AA, Veriava Y, Milne FJ. Plasma vitamin E levels in a chronically hemolyzing group of dialysis patients. Clin. Nephrol. 1986;25:42–47. [94] Cristol JP, Bosc JY, Badiou S, Leblanc M, Lorrho R, Descomps B, Canaud B. Erythropoietin and oxidative stress in haemodialysis: beneficial effects of vitamin E supplementation. Nephrol. Dial Transplant. 1997;12:2312–2317.

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[95] Roob JM, Khoschsorur G, Tiran A, Horina JH, Holzer H, Winklhofer-Roob BM. Vitamin E attenuates oxidative stress induced by intravenous iron in patients on hemodialysis. J. Am. Soc. Nephrol. 2000;11:539–549. [96] Islam KN, O’Byrne D, Devaraj S, Palmer B, Grundy SM, Jialal I. Alphatocopherol supplementation decreases the oxidative susceptibility of LDL in renal failure patients on dialysis therapy. Atherosclerosis. 2000;150:217–224. [97] Lassnigg A, Punz A, Barker R, Keznickl P, Manhart N, Roth E, Hiesmayr M. Influence of intravenous vitamin E supplementation in cardiac surgery on oxidative stress: a double-blinded, randomized, controlled study. Br. J. Anaesth. 2003;90:148–154. [98] Tarng DC, Huang TP, Chen TW, Yang WC. Erythropoietin hyporesponsiveness: from iron deficiency to iron overload. Kidney Int. 1999;69:S107–S118. [99] Podmore ID, Griffiths HR, Herbert KE, Mistry N, Mistry P, Lunec J. Vitamin C exhibits pro-oxidant properties. Nature. 1998;392:559. [100] Levine M, Daruwala RC, Park JB, Rumsey SC, Wang Y. Does vitamin C have a prooxidant effect? Nature. 1998;395:231. [101] Miyazaki H, Matsuoka H, Itabe H, Usui M, Ueda S, Okuda S, Imaizumi T. Hemodialysis impairs endothelial function via oxidative stress: effects of vitamin Ecoated dialyzer. Circulation. 2000;101:1002–1006. [102] Feldman L, Efrati S, Eviatar E, Abramsohn R, Yarovoy I, Gersch E, Averbukh Z, Weissgarten J. Gentamicin-induced ototoxicity in hemodialysis patients is ameliorated by N-acetylcysteine. Kidney Int. 2007;72:359–363. [103] Mazzon E, Britti D, De Sarro A, Caputi AP, Cuzzocrea S. Effect of N-acetylcysteine on gentamicin-mediated nephropathy in rats. Eur. J. Pharmacol. 2001;424:75–83. [104] Ajith TA, Usha S, Nivitha V. Ascorbic acid and alpha-tocopherol protect anticancer drug cisplatin induced nephrotoxicity in mice: a comparative study. Clin. Chim. Acta. 2007;375:82–86. [105] Lynch ED, Gu R, Pierce C, Kil J. Reduction of acute cisplatin ototoxicity and nephrotoxicity in rats by oral administration of allopurinol and ebselen. Hear Res. 2005;201:81–89. [106] Capizzi RL. Amifostine reduces the incidence of cumulative nephrotoxicity from cisplatin: laboratory and clinical aspects. Semin. Oncol. 1999;26:72–81. [107] Zurovsky Y, Haber C. Antioxidants attenuate endotoxin-gentamicin induced acute renal failure in rats. Scand. J. Urol. Nephrol. 1995;29:147–154. [108] Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Am. J. Kidney Dis. 1997;29:465–477. [109] Rodrigo R, Bosco C, Herrera P, Rivera G. Amelioration of myoglobinuric renal damage in rats by chronic exposure to flavonol-rich red wine. Nephrol. Dial. Transplant. 2004;19:2237–2244. [110] Giovannini L, Migliori M, Longoni BM, Das DK, Bertelli AA, Panichi V, Filippi C, Bertelli A. Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. J. Cardiovasc. Pharmacol. 2001;37:262-270. [111] Nath KA, Balla G, Vercellotti GM, Balla J, Jacob HS, Levitt MD, Rosenberg ME. Induction of heme oxygenase is a rapid protective response in rhabdomyolysis in the rat. J. Clin. Invest. 1992;90:267–270.

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[112] Onem Y, Ipcioglu OM, Haholu A, Sen H, Aydinoz S, Suleymanoglu S, Bilgi O, Akyol I. Posttreatment with aminoguanidine attenuates renal ischemia/reperfusion injury in rats. Ren. Fail. 2009;31:50-53.

In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter VI

Pre-Eclampsia Mauro Parra Fetal Medicine Unit, Obstetrics and Gynecology Department, University of Chile Clinical Hospital. Supported by FONDECYT, grant 1070948

Abstract Pre-eclampsia (PE) is the most important complication of human pregnancy worldwide and a major contributor to maternal and fetal morbidity and mortality. It is a disease of two stages. The first stage concerns the relative failure of early trophoblast invasion and remodeling of the spiral arteries, leading to a poor blood supply to the fetoplacental unit, exposing it to oxidative stress. The second stage is characterized by maternal endothelial dysfunction, leading to the clinically recognized symptoms of the syndrome, which include hypertension, proteinuria, thrombocytopenia and impaired liver function. Furthermore, the modification of spiral arteries occurs during the first and early second trimester of pregnancy, leading to uteroplacental hypoperfusion and fetal hypoxia. Despite much work in the last decade, the causes that trigger PE are uncertain and the predictive value of potential risk factors is poor. Increasing evidence suggests that placental and systemic oxidative stress plays a crucial role in its development. Indeed, oxidative stress and disrupting angiogenesis is considered the link bridging the two stages of the disease. Markers of oxidative stress in women with established PE have shown both increased lipid peroxidation in placental tissue, along with increased in maternal plasma biomarkers indicating decreased antioxidant capacity and increased lipid peroxidation. These findings have contributed to the interest in using antioxidants to prevent the development of PE. The lack of appropriate early predictors of the disease has determined that the risk groups for primary prevention of PE should be characterized on the basis of the clinical history of the patients and from knowing that is possible to establish some risk factors. A large number of publications suggest a potential role of antioxidant nutrients in the prevention of PE in women at high increased risk of the disease. Vitamins C and E have been the main antioxidants agents used for this purpose. Despite the biological properties of these compounds, exerting ROS scavenging and a

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down-regulation of ROS, the results of clinical trials do not support benefits for routine supplementation with vitamins C and E during pregnancy to reduce the risk of PE. This chapter examines the role of oxidative stress in the pathophysiology of PE and reviews the available data on the use of antioxidant compounds, mainly vitamins C and E, to prevent the development of this disease.

1. Introduction 1.1. Definition and Classification For the past hundred years, pre-eclampsia (PE) has been considered a placental pathology and its clinical management has remained practically unchanged since then [1] PE is a leading cause of maternal mortality in developed countries [2], and is associated with an increased rate of perinatal morbidity due to iatrogenic deliveries. Pre-eclampsia classically defined as a clinical syndrome of still unknown cause that develops in a previously normotensive woman after the second half of pregnancy, and it is characterized by an increased blood pressure (140/90 mmHg) and proteinuria greater than 300 mg in a 24 hour urine collection. Both blood pressure and proteinuria are resolved after delivery of the fetus [3]. The incidence of PE is about 5% of all pregnancies, and in about 20% of cases early onset PE leads to delivery before 34 weeks [4]. The pathology is more common in conditions as follows: primagravid women, maternal age above 40 years and multiparous women with change of partner, increased body mass index and obesity, previous history of pre-eclampsia, antiphospholipid antibodies, pre-existing diabetes, and multiple pregnancies [5-10]. Hypertension in pregnancy can be classified in four categories : a) pre-existing hypertension (3-5% of pregnancies), characterized by being present before pregnancy or diagnosed before 20 weeks of gestation; b) pregnancy-associated hypertension (12% of pregnancies) as appearance of high blood pressure after 20th weeks of gestation, which in turn can be sub-classified according to the presence of proteinuria in pre-eclampsia (5-6%) and gestational hypertension (6-7%); c) superimposed pre-eclampsia (25% of women with preexisting hypertension); and d) eclampsia when convulsion is present in a pregnant women with, or who later develop, hypertension [11].

1.2. Clinical Assessment of Pre-Eclampsia Normal pregnancy is characterized by a fall in blood pressure due to peripheral vasodilatation during the second trimester, and increased cardiac output and blood volume by about 50%. By contrast, severe PE has usually been associated with a low cardiac output and a high peripheral resistance, although some authors have reported contradictories results [12, 13]. Increased proteinuria observed in PE is characterized by loss of serum proteins and increase in capillary endothelial permeability. The decrease in blood volume leads to an increase in tissue edema.

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This change in blood volume is expressed through an increase in maternal hemoglobin concentration and increase risk of fetal growth restriction [14].Platelet count is reduced in normal pregnancy (<200x109/L) due to the physiological maternal blood-volume expansion, however, in severe PE cases there is a further progressive fall in platelet count as a result of both increased consumption and intravascular destruction [14]. In normal pregnancy, there is a raise in creatinine clearance with a concomitant decrease in serum creatinine and urea concentrations. In severe PE, serum creatinine tends to increase and it can be related with poorer outcome [15] The uric acid levels drop in normal pregnancy as an expression of increased renal excretion. In PE, on the other hand, there can be an increase in uric acid levels, correlating with poorer maternal and perinatal outcome. It is thought that this uric acid rise is due to either a decrease in renal function or the occurrence oxidative stress [16]. Finally, the liver may be damaged in severe cases of PE, associated with upper epigastric pain, distension and even rupture of the capsule into the peritoneal cavity. Altered liver enzyme, alanine and aspartate aminotransferase activities can also be seen as part of a characteristic severe condition called HELLP syndrome (hemolysis, elevated liver enzyme and low platelet) [16] With regards to the central nervous system, it can be also affected in PE, especially in severe conditions, such as stroke and eclampsia. Fortunately, in the last ten years these complications have been reduced due to improvement in the management of these cases introducing early delivery, antihypertensive drugs and magnesium sulphate [17].

2. Pathophysiology of Pre-Eclampsia The syndrome of PE involves several organ systems including placenta; kidney, liver, brain, the vasculature, the hematopoietic and coagulation system. Measurement of specific markers for each of these systems may not only indicate organ involvement before manifestation of the full maternal syndrome, but indicate that there are several different causes and presentations leading to PE. With certainty, the etiology of PE is still unknown. However, PE is characterized by certain pathophysiological, hematological and biochemical changes which some of them may be consequences or causes of this syndrome. The proposed sequence of events comprises endothelial dysfunction, defective trophoblast invasion, and consequential impaired placental perfusion, immune maladaptation and inflammation. The common link between these could be enhanced oxidative stress by excessive production of reactive oxygen species coupled with inadequate or overwhelmed antioxidant defense mechanisms. Pre-eclampsia is nowadays considered to be a syndrome derived from multiple mechanisms that contribute to the pathophysiology of this complex obstetric condition. There are several hypotheses, although abnormal placentation and endothelial dysfunction are almost always part of the explanations. The placental disease is represented by an abnormal extravillous trophoblast invasion of the spiral arteries, leading to placental hypoperfusion and/or ischemia/reperfusion cycles, which produce an oxidative stress state and consequently an increase in the reactive oxygen species (ROS) at the intervillous space. The placental oxidative stress through cytokine and chemokine adhesion

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molecules recruits and activates leukocytes at the intervillous space causing damage to the endothelial cells. ROS also increase lipid peroxidation and peroxynitration, leading to a reduction in nitric oxide and prostacyclin bioavailability and DNA oxidation. PE is also associated with hyperhomocysteinemia, which may increase ROS by reducing nitric oxide production through increasing asymmetric dimethyl-arginine (ADMA) which competes directly with L-arginine for eNOS. Together, as outlined in figure 6-1, these changes lead to the development of all the features of pre-eclampsia such as hypertension, edema, proteinuria and hypercoagulability. The linkage between placental hypoxia and maternal vascular dysfunction has been proposed to be via placental syncytiotrophoblast basement membranes shed by the placenta or via angiogenic factors which include soluble flt1 and endoglin secreted by the placenta that bind VEGF and PLGF in the maternal circulation. In PE, there is abundant evidence of altered reactivity of the maternal and placental vasculature and an altered production of autacoids [18].

Figure 6-1. An schematic diagram illustrating the proposed role of oxidative stress and the main contributory factors involving endothelial dysfunction, featuring clinical aspects of pre-eclampsia. ROS, reactive oxygen species; LDL, low density lipoproteins; Hcy, homocysteinemia; OX-LDL, oxidized LDL, NF-кB, nuclear factor kappa B; iNOS, inducible nitric oxide synthase; ADMA, asymmetric dimethyl-arginine; eNOS, endothelial nitric oxide synthase; VEGF, vascular endothelial growth factor; VWF, von Willebrand factor; TM, thrombomodulin; PGI2, prostacyclin; TXA2, thromoboxane A2, BH4, tetrahydrobiopterin.

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2.1. Placental Insufficiency Pre-eclampsia occurs only in the presence of the placenta and its resolution begins with the removal of the placenta. More than 70 years ago, E. W. Page suggested that the feature that characterized the preeclamptic placenta was its exposure to decreased perfusion [19]. It is accepted today that during early pregnancy, normal placentation occurs in a relatively hypoxic environment (<2% oxygen) that is essential for acceptable development, which in turn prevents trophoblast differentiation to an invasive phenotype [20-22] Intervillous blood flow increases at around 10-12 weeks of gestation and results in exposure of the trophoblast to increased oxygen tension (pO2) and increases in the activity and levels of the major antioxidant enzymes in the villous tissues, superoxide dismutase, catalase, and glutathione peroxidase [23]. Hypoxia inducible factor-1 (HIF-1), and specifically its 1α subunit expression, and transforming growth factor β3 (TGFβ3), an inhibitor of early trophoblast differentiation, mediates the effects of low oxygen tension during the first trimester of pregnancy. The expression of both molecules is high in early pregnancy and falls at around 10-12 weeks of gestation when placental pO2 levels are believed to increase due to unplug uterine spiral arteries [20] In addition, there is also evidence that leptin, TGFβ1 and PAI-2 are involved in the process of physiological trophoblast invasion [24- 26]. In normal pregnancy, progressive endovascular trophoblast invasion occurs progressively from the decidua into the inner third of the myometrium between 12 to 20 weeks of gestation [27- 29]. These changes are characterized by spiral artery remodeling including the disintegration of the tunica media along with the internal elastic lamina, and replacement of the endothelium with extravillous trophoblast cells expressing an endothelial phenotype. The changes involve conversion of the narrow muscular arterial tubes into flaccid and wider tubes that facilitate unimpeded placental perfusion with maternal blood flow required for adequate exchange of key molecules between the maternal and fetal circulations. As a consequence of previous physiological changes, the spiral arteries remodeling facilitates the ten-fold increase in uteroplacental blood flow that occurs between conception and term. Histological studies have shown that the process of spiral artery vascular remodeling is partial in pregnancies affected by PE or fetal growth restriction (FGR) [30]. Extravillous trophoblast invasion and spiral artery remodeling occur either very superficially or not at all in PE. This dysfunctional process of invasion and replacement of endothelial function results in both high resistance to flow in the maternal uterine arteries and relative placental hypoperfusion at a time period in which PE first symptoms may appear. One of the molecular explanations for an abnormal placentation was made by Cannigia et al. [31] who found that TGFβ3 expression was increased in human PE placentae when compared to age-matched controls and that inhibition of TGFβ3 by antibodies restored the invasive ability to the trophoblast cells in PE explants. The authors speculate that if oxygen tension fails to increase, or trophoblast does not detect this increase, expression of the two factors remains high, resulting in shallow trophoblast invasion and predisposing the pregnancy to PE. However, it is well known that abnormal placentation does not mean necessary PE and it is therefore necessary to find a link between the placental and maternal disease. Firstly,

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Roberts et al. [32] proposed a hypothesis about the pathophysiology of PE called the “two stage model of pre-eclampsia” which intend to resolve this dilemma. The first stage of this model, whose histological and molecular changes have been described above, is the reduction of maternal blood flow to the intervillous space. The second step of this model is the transference of the reduction of placental perfusion to systemic maternal pathophysiology. This abnormal perfusion of the intervillous space may lead to the production of different molecules which finally affect endothelial function and reduce organ perfusion. The search for this factor has led to the identification of numerous substances as candidates to be the “Factor X”. More recently, there is also an integrated model proposed by Redman et al, which assumes that the placenta plays a central role in the pathogenesis of PE, but that abnormal placentation is unlikely to be the exclusive cause of this syndrome [33]. The authors explain that a normal pregnancy is associated with an apoptotic physiological export of syncytiotrophoblast microparticles into the maternal circulation, leading to a systemic inflammatory response [34]. The hypothesis suggests that PE may be explained by either the presence of a larger or oxidatively stressed placenta [35]. A larger placenta is also seen in gestational diabetes, twin pregnancy, term or molar pregnancies. An oxidatively stressed placenta exacerbates the already established inflammatory state in normal pregnancies, leading therefore to PE.

2.2. Oxidative Stress in Pre-Eclampsia Although the cause of PE still remains unknown, it has been proposed that enhanced oxidative stress is a basic component of this condition that could provide the connection between abnormal placentation and the maternal syndrome [36, 37]. The main source of reactive oxygen species (ROS) initiating the pathophysiological events appears to be the placenta [38] but maternal leukocytes and maternal endothelium are also likely contributors. The failure of placental perfusion, explained previously, is likely to result in hypoxia-reoxygenation cycles which lead to oxidative stress due to changes in the vascular vasomotor activity by maternal humoral and neural influences [39]. It has been recently suggested that the cause of PE is the abnormal oxidative insult associated with preeclampsia and not the mRNA expression of antioxidant proteins that may be responsible for reduced antioxidant enzyme activity in preeclamptic placenta [40]. Other mechanisms of ROS production in PE pregnancies should be the activation of maternal neutrophils by syncytotrophoblast microvesicles following deportation due to increased apoptotic or aponecrotic mechanisms locally activated during the passage of maternal blood through the placenta [41]. Thus, isolated neutrophils from women with PE synthesize more superoxide than those of normotensive pregnant women [42]. Consecutively, activated neutrophils may contribute to the activation of the vascular endothelium, contributing therefore to the pathophysiology of PE [43]. It was found that oxidative stress early in pregnancy influenced pregnancy outcome, as assessed by the finding that urinary F2isoprostanes, biomarkers of lipid peroxidation, are associated with an increased risk of preeclampsia and a decreased proportion of female births [44]. Recent studies have suggested

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that this imbalance between oxidant and antioxidant is the effect of disease and not the causative factor [45]. 2.2.1. Placental Ischemia-Reperfusion and Reactive Oxygen Species Despite the possibility that the generation of the placental oxidative stress may be related to hypoxia due to reduced uteroplacental perfusion, hypoxia itself is not sufficient to account for all the morphological findings [46]. In fact, an ischemia-reperfusion cycle at the uteroplacental blood flow may be a more important factor for establishing placental oxidative stress [47]. As a consequence, an increased capacity of placental, as well as of other cells, to generate ROS has been found in PE [38, 48]. Oxidative stress in this context is a consequence of an imbalance between excessive generation of ROS and reduced capacity of the antioxidant defenses, causing placental damage, which in turn could account for the increased rates of infarction and syncytial necrosis observed in this condition. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic conditions [49]. 2.2.2. Pro-Oxidant Enzymes The most common ROS is the superoxide anion. Xanthine and NADPH oxidases have been identified as major vascular superoxide-forming enzyme systems, but the contribution of xanthine oxidase (XO) is generally minor [50]. However, XO is abundantly expressed in cytotrophoblast and syncytiotrophoblast cells due to the fact that ischemia-reperfusion is a potent stimulus to the conversion from xanthine dehydrogenase [48, 51]. In addition, NADPH oxidase activity, constitutively observed in the trophoblast of the human placenta, is highly stimulated in PE [52]. This enzyme consists mainly of 5 subunits and it has been observed that placentas from preeclamptic patients show increased expression of NADPH oxidase components p22, p47, and p67 [53]. Other forms of NADPH oxidase are also implicated in the pathophysiology of PE, such as those present in phagocytes (neutrophilic and eosinophilic granulocytes, monocytes, and macrophages) and vascular cells. NADPH oxidase mediates increased isolated neutrophils production of superoxide observed in women with PE [54]. It been well established that vascular NADPH oxidase plays a major role in the development of hypertension [55] and is a target for a down-regulation exerted by antioxidant vitamins. 2.2.3. Biomarkers of Oxidative Stress As we have reviewed, PE is associated with an exaggerated production of superoxide anion via pro-oxidant enzyme generated due to ischemia-reoxygenation cycle in placenta and activated leukocytes and endothelial cells. Oxidative stress state can produce lipid peroxidation, protein carbonylation and DNA alteration in different feto-maternal tissues. The main biomarkers of oxidative stress are summarized below. Peroxynitrite: the increased generation of superoxide anion by the placenta, activated leukocytes, or endothelial cells is accompanied by an up-regulation of inducible nitric oxide synthase (iNOS), an isoform mainly located in macrophages and neutrophils, leading to increased formation of peroxynitrite. In fact, the rate constant for the formation of

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peroxynitrite is 3 times faster than that of the interaction of superoxide with superoxide dismutase (SOD) [56]. Malondialdehyde (MDA): peroxynitrite interacts with lipids leading to peroxidation and MDA and conjugated diene formation [57]. In PE pregnancies, MDA levels are reported to be elevated in maternal plasma and placental tissue [58, 59] as well as in erythrocytes, and the severity of the disease correlates with the MDA concentration in both the serum [38] and in erythrocytes [60]. Our own data also showed that there was significantly increased MDA placental production from PE patients compared to normal control group [61]. Additionally, there was a direct correlation between the lipid peroxidation level and the PE severity [62]. Therefore, PE can be associated with increased lipid peroxidation. It is also remarkable that MDA products may behave as toxic bifunctional electrophiles, due to reactivity with proteins, phospholipids, and DNA, generating stable products at the end of a series of reactions to form propane adducts [63]. F2-isoprostane: this molecule is a product of lipid peroxidation which is formed by a nonenzymatic peroxidation of arachidonic acid. This compound serves as an index of lipid peroxidation in several diseases, including PE [64, 65]. The production and secretion rates of F2-isoprostanes by placentas obtained from women with PE were significantly higher than those of controls [66], providing convincing evidence that oxidative stress and lipid peroxidation are abnormally increased in preeclamptic placenta. Carbonyls: the direct damage of proteins during oxidative stress can give rise to the formation of protein carbonyls, which may serve as biomarkers for general oxidative stress. Higher levels of protein carbonyls in both the placenta and decidua were found in women with mild to severe PE either alone [67] or with concurrent HELLP syndrome [68]. In addition, our data showed that PE women were characterized by alteration of all oxidative stress parameters, i.e. there was a significant reduction in total antioxidant capacity of plasma (FRAP, ferric reducing ability of plasma), increased uric acid, F2- isoprostane, and carbonyl plasma levels [61]. 2.2.4. Antioxidant Defense System in Pre-Eclampsia Antioxidant enzymes: physiologically by 10 to 12 weeks of gestation, the onset of blood flow throughout the modified spiral arteries results in an increased oxygen tension and simultaneous elevation in the expression and activity of the antioxidant enzymes [21]. On the contrary, the increased concentration of superoxide in the PE placental tissue [69] was found to be associated with decreased SOD activity and mRNA expression for CuZn-SOD in trophoblast cells isolated from PE placentas [70]. Also, it was found that these women show a decrease in plasma levels of SOD [59]. Another study reports reduced catalase and SOD activities, with a concomitant elevation of lipid peroxidation products [71]. With regards to total glutathione peroxidase in placenta from PE women, there are contradictory data, since one study has found increased levels [71] and other reported a decreased activity in placenta from PE women [72]. It was found that SOD and catalase activity in PE placental tissues lower than in the control group [61]. Nonenzymatic antioxidants: natural or endogenous antioxidants measured in maternal blood, placenta, and deciduas are reduced in PE women [67, 73-76] however, they do no differ between mild or severe conditions [77]. A large number of studies agree that PE is

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associated with a decrease in serum levels of ascorbic acid (vitamin C) and α-tocopherol (vitamin E) [78, 79] as well as a decrease of other lipid soluble antioxidants such as coenzyme Q10 [80], carotenoids, and retinol [81]. Other studies have reported that there is no significant difference in plasma vitamin E concentration, when PE pregnancies are compared to controls [82]. 2.2.5. Oxidative Stress Biomarkers as Predictors of Pre-Eclampsia In general terms screening tests are available to let a pregnant woman know about prevention for PE at any one of three different stages: primary, secondary, or tertiary. The concept of primary prevention is conceived to avoid the occurrence of a disease. Secondary prevention, in the context of PE, implies inhibiting the disease process before appearance of clinically recognizable disease. Finally, tertiary prevention, or treatment, means prevention of complications caused by the disease process. For the purpose of this chapter, we will only review data available on screening test for primary and secondary prevention. Primary prevention of PE should be determined by the clinical history of the patients and from knowing that it is possible to establish some risk factors. The most significant factors to be considered in the patient medical files are: the history of PE, antiphospholipid antibodies, pre-existing diabetes and body mass index above 35 [8-10]. However, all these variables are only able to predict just 30% of appearance of PE [83]. Several demographic factors have been associated with the risk of developing PE during pregnancy. Those mentioned above are the most relevant to be used as screening for this condition. Secondary prevention of a disease is only possible if the following three requirements are met: knowledge of pathophysiological mechanisms, availability of methods of early detection, and resources for intervention and correction of the pathophysiological changes. Although the pathogenesis of PE is poorly understood, the high resistance placental circulation shown on uterine artery Doppler study during the second trimester is often used as a predictor of PE. It has been reported recently that the very high likelihood ratio of PE in the abnormal uterine artery Doppler screen-positive group confirms the reliability of this secondtrimester screening test in diagnosing impaired placentation [61] and the findings are compatible with those of other previous screening studies during the first [84] and second trimester [85-87]. On the other hand, PE pregnancies have been associated with increased PAI-1/PAI-2 ratio, sFlt-1, F2-isoprostane and reduced free PlGF at 23 weeks of gestation, suggesting that impaired placentation, anti-angiogenic mediators, endothelial dysfunction, and oxidative stress are the factors that predate the development of PE by several weeks. The findings that PE women show increased plasma concentration of 8-epi-prostaglandin F2α supports the hypothesis that poor uteroplacental perfusion predisposes to an increase in placental free radical synthesis and, thereby, to maternal oxidative stress. Our results are in agreement with those of Chappell et al who showed that this marker of lipid peroxidation was increased in women who later developed PE [88]. Other biochemical markers that have been significantly associated with the appearance of PE are endothelial dysfunction and anti-vasculogenesis biochemical markers. It has been

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found that PAI-1/PAI-2 ratio is significantly increased in women who later develop PE. This is in agreement with other authors [88] and with the hypothesis that endothelial dysfunction is the main feature of this condition and predates its onset [1, 89]. It has been observed increased circulating anti-angiogenic factor (sFlt1) and reduced free PlGF in women who subsequently developed PE, results in agreement with previous publications [90, 91]. As our data also show, Levine et al. [91] have demonstrated that alterations of the sFlt1 appeared to be greater in women who had early-onset PE and in women with PE who delivered a small for gestational age infant, suggesting that defective angiogenesis may be especially important in these cases. However, these biochemical markers appear to be a consequence rather than a cause of this disease, because they do not improve second trimester uterine artery Doppler detection rate Although second trimester screening test, either combined markers or uterine artery Doppler alone, has demonstrated to reach a good detection rate for early-onset PE, all prevention trial performed till today had failed to demonstrate any benefit in this high-risk group. Consequently, recent publications have shown that a combined test between clinical, biochemical and uterine artery Doppler during the first trimester of pregnancy can predict about 80% of early-onset PE (<34 weeks of gestation) with only 10% false positive rate [83]. These publications are in agreement with our own data, showing that around 70% of early-onset PE can be predicted correctly at 12 weeks using a combined model of uterine artery Doppler, placental growth factor and body mass index. Oxidative stress markers, such as F2- isoprostane, MDA, FRAP and uric acid plasma levels were not significantly different to the control group [61]. In addition, the urinary excretion of F2-isoprostanes was associated with an increased risk of PE [ 44 ]. However, a recent gene expression study performed in chorionic villous samples obtained at 11 weeks of pregnancy from 5 women who later develop PE, have demonstrated that mRNA expressions of SOD and other markers involved in the development of normal placentation were significantly altered compared to matched-control group [92]. Other biochemical markers testing during the first trimester of pregnancy with high rate of detection rate for early-onset PE are placental protein 13 [93] and pregnancy-associated plasma protein [94]. Plasma levels of these molecules are lower than controls at 11-14 weeks of gestation. Both markers might play a role in the process of extravillous trophoblast invasion and spiral artery remodeling [95, 96].

3. Prevention with Antioxidants To date, many strategies aimed at secondary prevention of PE have been studied, although none of them has proved to prevent the clinical appearance of PE [11, 97, 98 ]. It is accepted that free radicals are promoting maternal vascular dysfunction and PE in women are associated with an oxidative stress state, which is characterized by increased markers of lipid peroxidation, such as MDA [ 64 ] and F2-isoprostane [99] and reduced plasma and placental levels of antioxidants [100, 101]. Therefore, the possibility of implementing an antioxidant therapy with vitamins C and E, based on the multiple biological properties of these agents in addition to prevention of lipid peroxidation, appears to be quite

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well supported [102]. These defense mechanisms, involving antioxidant vitamins and enzyme systems, may restrain the extent of damage caused by oxidative stress [103]. On this line, vitamins C and E down-regulates NADPH oxidase, which is the major source of superoxide anion, at the vascular wall level. Regulation on this enzyme, as reviewed previously, could play a key role as mediator in the development of systemic pathological processes such as impairment of endothelium-dependent vasodilatation [104], inflammation and increased platelet aggregation. In addition, there are also data in animal models, such as pig coronary artery and aorta from spontaneously hypertensive rats, demonstrating that vitamins C and E may cause down-regulation of NADPH oxidase and up-regulation of eNOS [105]. Although the exact mechanisms whereby antioxidant vitamins act on those enzymes are unclear, five possible mechanisms have been suggested: a) regulating protein expression of the NADPH oxidase at the transcriptional or post-translational levels [106]; b) inhibiting or interrupting the complex formation of the NADPH oxidase subunit at cell membrane [107]; c) preventing p47phox NADPH oxidase subunit membrane translocation and phosphorylation [108]; d) stimulating eNOS activity at endothelial cells by increasing the intracellular availability of the eNOS cofactor tetrahydrobiopterin (BH4) that would further increase NO synthesis [109, 110]; e) inhibiting the up-regulation of ICAM-1 [111] and increased production of IL-6 [112] which might be mediated by NF-κB activation in PE. It has been suggested that the deleterious effect of ROS may be counteracted by an antioxidant therapy, and that more studies are necessary to determine the optimum dosing and timing of antioxidant administration, since an inappropriate antioxidant treatment in pregnant women may have deleterious consequences, reducing placental cells proliferation until to cell death [113]. Although vitamins C and E inhibit apoptosis of cultured human term placenta trophoblast [114], it was reported that exposure of these cells to high levels of antioxidant vitamins C and E may affect placental function, in terms of decreasing secretion of hCG, placental immunity and increasing production of TNF-α. Such alterations are known to lead to endothelial dysfunction and adverse pregnancy outcomes, such as fetal growth restriction [115]. Chappell et al. [87] have carried out a randomized clinical trial using supplementation with vitamins C and E in a high risk group of pregnant women during the second half of pregnancy. They have shown that antioxidant vitamins had beneficial effects on biochemical markers of the disease, and may be beneficial in the prevention of clinical PE, although the latter was not statistically significant. Recently, there have been published four randomized trials regarding the effect of antioxidant vitamins in preventing PE in high and low risk women [115-118]. Beazley et al. [115] planned to randomize 220 high-risk women, but they stop this study due to lack of funding. Finally, they reported their result base on a group of only 100 women who were given antioxidant vitamins from 14 weeks onwards. They did not find any significant difference in the rate of PE between both groups. Poston et al. [117] randomized 2410 high-risk women who were recruited between 14 to 22 weeks of gestation according clinical history and current pregnancy. They found that there was no reduction in the incidence of PE or fetal growth restriction in women who received vitamin E and C. They also raised a concern about using antioxidant vitamins in pregnant women because it was associated with increased severity of hypertension. The next study was performed in 1877 nulliparous women who were recruited between 14 and 22 weeks of gestation. They found

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that there were no significant differences between the vitamin and placebo groups in the risk of PE and other serious maternal or newborn outcomes [118]. Furthermore, a clinical trial conducting in 707 high-risk Brazilian women recruited between 12 to 20 weeks of gestation were in agreement with the three previous publications that incidence of PE was not modified with antioxidant vitamins. Finally, a recent meta-analysis involving 6533 pregnant women corroborates the previous data that antioxidant vitamins did not reduce the relative risk of PE and other adverse outcomes [119]. However, antioxidant vitamins did significantly increase antihypertensive therapy (77%) and antenatal hospital admission for hypertension (54%). They conclude that evidence does not support routine antioxidant supplementation during pregnancy to reduce the risk of pre-eclampsia and called attention on possible serious complication in pregnancy. At present, the results of these studies have been mainly disappointing and controversial, but they should be interpreted with caution. In fact, most studies were done in high-risk population with extremely diverse pathophysiological backgrounds and also supplementation was started mostly after 14 weeks of gestation, a time at which physiological placentation has been already established. As it has been explained in other section of this reviewed, it could be hypothesized that the derangement of placentation is a consequence of earlier events during gestation, based on the normal trophoblast invasion occurring in a way temporally and spatially unique that involves both degradative and adhesive interactions [1]. Many of these adaptive changes appear to be disrupted in PE due to decreased degradative ability secondary to lower levels or cytokine inactivation of MMP-9, [120, 121] or improper expression of adhesion molecules [29], all processes triggered by oxidative stress. Recently, placental oxidative stress has been attributed to increased expression of NADPH oxidase [122] and ET-1 [123] in the placenta, a process occurring very early during placental development, likely accounting for the impaired trophoblast migration and its consequences (figure 6-2). This view could explain the failure of the clinical trials to prevent the development of PE through later antioxidant therapy. Therefore, it is necessary to improve our capacity to develop earlier predictive test based on uterine artery Doppler in combination with clinical history and biochemical markers, to detect a high-risk group destine to develop early-onset or severe PE [124]. Endothelial cell apoptosis of the spiral arteries is postulated as the mechanism by which trophoblast influences vessel remodeling. There are several mechanisms which could activate apoptosis in vascular cells, one of them being the interaction between Fas/FasL. Furthermore, it has recently been shown that first trimester trophoblast cells can secrete FasL [125, 126]. Candidates that should be studied for their involvement in the regulation of endothelial apoptosis by the trophoblast cells include intervillous space levels of oxygen during the first trimester (hypoperfusion/reperfusion state associated with oxidative stress state), activation of HIF-1, and the role of L-arginine/nitric oxide synthesis in trophoblast invasion.

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Figure 6-2. Hypothesis to explain the contribution of oxidative stress in the pathogenesis of the syndrome of pre-eclampsia. The counteracting effect of antioxidant vitamins C and E in two crucial stages of the process of placentation is indicated by the symbol ( ). ROS, reactive oxygen species; MMP-9, matrix metaloproteinase-9; VEGF, vascular endothelial growth factor. (Adapted from Rodrigo et al. Fundam Clin Pharmacol 2007; 21:111-127).

It is also accepted that cytotrophoblast undertakes a program of pseudo-vasculogenesis during normal pregnancy and it has been postulated that cell-surface endoglin (Eng) may play a role in the regulation of this process. Cell-surface Eng inhibits trophoblast invasion and therefore it is speculated that increased soluble endoglin (sEng) observed in PE may be a compensatory response by the placenta to limit the effects of cell-surface Eng [127]. Thus, factors associated with vasculogenesis, such as sFlt, PlGF and sEng, have been suggested to

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be causally involved in the manifestation of PE. First of all, the authors showed that sEng and sFlt-1 can induce endothelial dysfunction in vitro, through inhibiting VEGF and TGF-β stimulation of endothelial-dependent NO activation. Secondly, treatment of pregnant rats with sFlt-1 and sEng induced signs of severe PE, including development of HELLP and fetal growth restriction. Interestingly, they showed that s-Flt-1, through decreasing activity of eNOS, is associated with increased vascular permeability in the maternal kidneys (increased proteinuria) and vasoconstriction, while, sEng, through inhibiting the antithrombotic factor prostacyclin, is associated with the procoagulant state and thrombocytopenia observed in this condition. Recent data suggest that in a pathophysiological condition, such as PE, the deleterious effect of ROS may be counteracted by an antioxidant therapy, and that there is an urgent need to investigate the optimum dosing and timing of antioxidants administration, since an inappropriate antioxidant treatment in pregnant women may have deleterious consequences by reducing placental cells proliferation to cell death.

4. Conclusions and Perspectives From the data reviewed we conclude that PE in women is characterized by oxidative stress, abnormal vasculogenesis and endothelial dysfunction. Furthermore, uterine artery Doppler characteristics proved to be the best predictor of PE during the second trimester, and were associated with markers of abnormal vasculogenesis. The findings observed in this chapter add support strength to the evidence implicating placental insufficiency and oxidative stress as critical events in the pathogenesis of PE, and add knowledge about the best way to predict these conditions using a combination between clinical, biochemical and uterine artery Doppler assessment during the first and second trimester of pregnancy. However, there is still lacking of data to promote the use of antioxidant vitamins as early prevention of this condition. This chapter strengthens the hypothesis that severe cases of PE, with or without fetal growth restriction, are explained by abnormal mechanisms of early placentation and vasculogenesis which involve oxidative stress and endothelial dysfunction as constitutive parts of the PE syndrome. Further investigation of the factors associated with the intriguing process of extravillous trophoblast invasion to spiral arteries during the first part of pregnancy is therefore needed. The alteration of this physiological process occurs during the first 20 weeks of pregnancy and is characterized by a transient coexistence between trophoblast, endothelial and vascular smooth muscle cells in the invaded spiral arteriesAlthough it is highly likely that the regulation of this fundamental physiological process is more complex than it is hypothesized, studies on the regulation of the extravillous trophoblast induction of endothelial apoptosis and abnormal vasculogenesis would be important for determining the failure of these processes to occur in PE. On the other hand, and following our hypothesis, it would be also interesting to study the interaction between the described poor placentation and its expression at the maternal level, such as endothelial dysfunction and haematological alterations.

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In summary, further studies on the biochemical alteration of the abnormal placentation observed in PE and its correlation with the systemic expression of pregnancy-induced hypertension are necessary for a better understanding of the pathophysiology of the PE syndrome, and associated conditions. It is also important to establish rational methods of PE screening, prevention and treatment.

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[51] Hassoun PM, Yu FS, Shedd AL, Zulueta JJ, Thannickal VJ, Lanzillo JJ, Fanburg BL. Regulation of endothelial cell xanthine dehydrogenase xanthine oxidase gene expression by oxygen tension. Am. J. Physiol. 1994;266:L163-171. [52] Matsubara S, Takizawa T, Takayama T, Izumi A, Watanabe T, Sato I. Immuno-electron microscopic localization of endothelial nitric oxide synthase in human placental terminal villous trophoblasts-normal and pre-eclamptic pregnancy. Placenta. 2001;22:782-786. [53] Dechend R, Viedt C, Müller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003;107:1632-1639. [54] Lee VM, Halligan AW, Ng LL. Neutrophil intracellular pH and Na+/H+ exchanger activity in pre-eclampsia. Metabolism. 2003;52:87-93. [55] Touyz RM, Yao G, Quinn MT, Pagano PJ, Schiffrin EL. p47phox associates with the cytoskeleton through cortactin in human vascular smooth muscle cells: role in NAD(P)H oxidase regulation by angiotensin II. Arterioscler Thromb. Vasc. Biol. 2005;25:512-518. [56] Huie RE, Padmaja S. The reaction of no with superoxide. Free Radic. Res. Commun. 1993;18:195-199. [57] Var A, Yildirim Y, Onur E, Kuscu NK, Uyanik BS, Goktalay K, Guvenc Y. Endothelial dysfunction in preeclampsia. Increased homocysteine and decreased nitric oxide levels. Gynecol. Obstet. Invest. 2003;56:221-224. [58] Madazli R, Benian A, Aydin S, Uzun H, Tolun N. The plasma and placental levels of malondialdehyde, glutathione and superoxide dismutase in pre-eclampsia. J. Obstet. Gynaecol. 2002;22:477-480. [59] Aydin S, Benian A, Madazli R, Uludag S, Uzun H, Kaya S. Plasma malondialdehyde, superoxide dismutase, sE-selectin, fibronectin, endothelin-1 and nitric oxide levels in women with preeclampsia. Eur. J. Obstet. Gynecol. Reprod. Biol. 2004;113:21-25. [60] Madazli R, Benian A, Gümüştaş K, Uzun H, Ocak V, Aksu F. Lipid peroxidation and antioxidants in preeclampsia. Eur. J. Obstet. Gynecol. Reprod. Biol. 1999;85:205-208. [61] Parra, M., Rodrigo, R., Barja, P., Bosco, C.,Fernández, V., Muñoz, H., Soto-Chacón, E. Screening test for preeclampsia through assessment of uteroplacental blood flow and biochemical markers of oxidative stress and endothelial dysfunction. Am. J. Obstet. Gynecol. 2005;193:1486–1491. [62] Panburana P, Phuapradit W, Puchaiwatananon O. Antioxidant nutrients and lipid peroxide levels in Thai preeclamptic pregnant women. J. Obstet. Gynaecol. Res. 2000;26:377-381. [63] Blair IA. Lipid hydroperoxide-mediated DNA damage. Exp. Gerontol. 2001;36:14731481. [64] Hubel CA, McLaughlin MK, Evans RW, Hauth BA, Sims CJ, Roberts JM. Fasting serum triglycerides, free fatty acids, and malondialdehyde are increased in preeclampsia, are positively correlated, and decrease within 48 hours post partum. Am. J. Obstet. Gynecol. 1996;174:975-982.

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[81] Williams MA, Woelk GB, King IB, Jenkins L, Mahomed K. Plasma carotenoids, retinol, tocopherols, and lipoproteins in preeclamptic and normotensive pregnant Zimbabwean women. Am. J. Hypertens. 2003;16:665-672. [82] Ben-Haroush A, Harell D, Hod M, Bardin R, Kaplan B, Orvieto R, Bar J. Plasma levels of vitamin E in pregnant women prior to the development of preeclampsia and other hypertensive complications. Gynecol. Obstet. Invest. 2002;54:26-30. [83] Plasencia W, Maiz N, Bonino S, Kaihura C, Nicolaides KH. Uterine artery Doppler at 11 + 0 to 13 + 6 weeks in the prediction of pre-eclampsia. Ultrasound Obstet. Gynecol. 2007;30:742-749. [84] Martin AM, Bindra R, Curcio P, Cicero S, Nicolaides KH. Screening for pre-eclampsia and fetal growth restriction by uterine artery Doppler at 11-14 weeks of gestation. Ultrasound Obstet. Gynecol. 2001;18:583-586. [85] Albaiges G, Missfelder-Lobos H, Lees C, Parra M, Nicolaides KH. One-stage screening for pregnancy complications by color Doppler assessment of the uterine arteries at 23 weeks' gestation. Obstet. Gynecol. 2000;96:559-564. [86] Papageorghiou AT, Yu CK, Bindra R, Pandis G, Nicolaides KH; Fetal Medicine Foundation Second Trimester Screening Group. Multicenter screening for preeclampsia and fetal growth restriction by transvaginal uterine artery Doppler at 23 weeks of gestation. Ultrasound Obstet. Gynecol. 2001;18:441-449. [87] Chappell LC, Seed PT, Briley AL, Kelly FJ, Lee R, Hunt BJ, Parmar K, Bewley SJ, Shennan AH, Steer PJ, Poston L. Effect of antioxidants on the occurrence of preeclampsia in women at increased risk: a randomised trial. Lancet. 1999;354:810-816. [88] Chappell LC, Seed PT, Briley A, Kelly FJ, Hunt BJ, Charnock-Jones DS, Mallet AI, Poston L. A longitudinal study of biochemical variables in women at risk of preeclampsia. Am. J. Obstet. Gynecol. 2002;187:127-136. [89] Redman CW, Sargent IL. Pre-eclampsia, the placenta and the maternal systemic inflammatory response--a review. Placenta. 2003;24 Suppl A:S21-S27. [90] Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Invest. 2003;111:649-658. [91] Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA. N. Engl. J. Med. 2004;350:672-683. [92] Farina A, Sekizawa A, De Sanctis P, Purwosunu Y, Okai T, Cha DH, Kang JH, Vicenzi C, Tempesta A, Wibowo N, Valvassori L, Rizzo N. Gene expression in chorionic villous samples at 11 weeks' gestation from women destined to develop preeclampsia. Prenat. Diagn. 2008;28:956-961. [93] Romero R, Kusanovic JP, Than NG, Erez O, Gotsch F, Espinoza J, Edwin S, Chefetz I, Gomez R, Nien JK, Sammar M, Pineles B, Hassan SS, Meiri H, Tal Y, Kuhnreich I, Papp Z, Cuckle HS. First-trimester maternal serum PP13 in the risk assessment for preeclampsia. Am. J. Obstet. Gynecol. 2008;199:122.e1-122.e11.

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In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter VII

Metabolic Syndrome Rodrigo Castillo Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile Supported by FONDECYT, grant 1070948

Abstract The biochemical steps linking insulin resistance with the metabolic syndrome have not been completely clarified. Mounted experimental and clinical evidence indicates that oxidative stress is an attractive candidate for a central pathogenic role since it potentially explains the appearance of all risk factors and supports the clinical manifestations. Indeed, metabolic syndrome patients exhibit activation of biochemical pathways leading to increased delivery of ROS, decreased antioxidant protection and increased lipid peroxidation. The described associations between increased abdominal fat storage, liver steatosis and systemic oxidative stress, the diminished concentration of nitric oxide derivatives and antioxidant vitamins, and the endothelial oxidative damages observed in subjects with the metabolic syndrome support oxidative stress as the common secondlevel event in an unifying pathogenic view. Moreover, it has been observed that oxidative stress regulates the expression of genes governing lipid and glucose metabolism through activation or inhibition of intracellular sensors. Diet constituents can modulate redox reactions and the oxidative stress extent, thus also acting on nuclear gene expression. As a consequence of the food–gene interaction, metabolic syndrome patients may express different disease features and extents according to the different pathways activated by oxidative stress-modulated effectors. This view could also explain family differences and interethnic variations in determining risk factor appearance.

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1. Introduction The metabolic syndrome is a multifactorial condition leading to accelerated atherosclerosis and increased risk for diabetes. It is associated with major cardiovascular events and a high mortality rate [1]. The metabolic syndrome is characterized by different combinations of three or more of the following features: abdominal obesity, blood hypertension, hyperglycemia and serum dyslipidemia as defined by the criteria of the Third Report of the National Cholesterol Education Program Adult Treatment Panel III [2] or by the updated criteria of the International Diabetes Federation [3]. Epidemiological surveys show that the metabolic syndrome is extremely common. The 1999–2002 National Health and Nutrition Examination Survey estimated the age-adjusted prevalence of the metabolic syndrome in U.S. adults over 20 years to be between 34.6% and 39.1% and to be even higher if considering adults over 60 years. It is a little more common in men, and also there exist ethnic differences. Overall, the prevalence of the metabolic syndrome parallels the increasing aging population and “epidemic” obesity [4]. The link between individual metabolic syndrome components is unknown. Recent studies support the view that these metabolic abnormalities [5] have a single factor may underlie the association, the insulin resistance [6]. The fact that insulin resistance and abdominal obesity are also associated with perturbations in plasma adipokine levels, altered fatty acid metabolism, endothelial dysfunction, procoagulant state and systemic inflammation underscores the breadth and complexity of the pathophysiology of this clustering [7]. Therefore, the identification of common basic mechanisms driving to a unifying pathogenic hypothesis for the metabolic syndrome would be helpful in explaining the clinical manifestations. In this point, oxidative stress could explain most of the second-level events resulting in risk factor appearance and may lead to a unitary pathogenic view of this chronic disorder. Oxidative stress has been associated with all the individual components and with the onset of cardiovascular complications in subjects with the metabolic syndrome [8, 9]. In a recent study [10, 11], the role of oxidative stress in the pathophysiologic interactions among the constituent factors of the metabolic syndrome has been remarked. Although some of the constituent characteristics of the metabolic syndrome are known to share common pathogenic mechanisms of damage, the impact of hereditary predisposition and the regulation of gene expression as well as the role of environment and dietary habit in determining inflammatory process-triggered oxidation are still unclear. These aspects of the problem deserve special attention since it is hypothesized that in patients with the metabolic syndrome, oxidative stress may be amplified by a concomitant antioxidant deficiency that may favour the propagation of oxidative alterations from intra- to extracellular spaces and from confined to distant sites, thus realizing a systemic oxidative stress state [12, 13]. Altogether, these considerations would suggest a unifying hypothesis to explain the mechanisms underlying the onset and development of metabolic syndrome-associated risk factors. As the following subsections report, excessive free radical production and oxidative damages are supported by several experimental demonstrations and human observations. Therefore, oxidative stress appears to possess the credentials to mechanistically explain the

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perpetuation of insulin resistance, the altered energy production, the endothelial dysfunction, and the appearance of vascular complications in this condition.

2. Pathophysiology of Metabolic Syndrome Metabolic syndrome is associated with insulin resistance. It is not a consequence of insulin resistance alone, but a direct consequence of the lack of insulin action. This is most evident in patients with insulin receptor mutations or autoimmune antibodies to the insulin receptor; they may have 100-fold or greater elevations of circulating insulin or require similarly high doses of exogenous insulin to control diabetes [14]. These patients exhibit a distinct syndrome with acanthosis nigricans and a high risk of diabetes, but typically have no obesity, hypertension, or atherogenic dyslipidemia [15]. Moreover, patients with type 1 diabetes mellitus, who lack insulin, do not exhibit the same atherogenic lipoprotein phenotype typical of patients with metabolic syndrome or type 2 diabetes mellitus. Lean type 1 diabetic mellitus patients do not characteristically have insulin resistance. If metabolic syndrome does not result purely from a lack of insulin effect, then how might insulin resistance generate other features of the syndrome? Proposed mechanisms center around 3 themes: effects of mild to moderate hyperglycemia, effects of compensatory hyperinsulinemia, and effects of unbalanced pathways of insulin action [16]. Hyperglycemia, largely postprandial and below diabetic levels, may lead to a variety of effects usually associated with diabetes. For example, moderate hyperglycemia might be postulated to cause accelerated atherogenesis via advanced glycosylated end products or via enhanced collagen formation [17]. (for more details see chapter 8). Another important mechanism may be the compensatory hyperinsulinemia. The maintenance of normal post-prandial glucose homeostasis requires that pancreatic beta cells secrete a normal amount of insulin in response to the hyperglycemic challenge, with resultant hyperinsulinemia [18]. Insulin stimulates the glucose uptake by muscle, which is the tissue responsible for the disposal of 80% to 90% of the ingested glucose load, and [2] suppresses endogenous glucose production, which is generated mainly in the liver. In insulin-resistant conditions, the ability of insulin to augment glucose uptake and inhibit hepatic glucose production is impaired. The resultant hyperglycemia presents a stimulus to the beta cells, which secrete large amounts of insulin after meals. Initially, attention was directed to the concept that certain organs and tissues can have lesser degrees of insulin resistance than skeletal muscle and liver [19]. For example, the high insulin concentration required to produce normal glucose uptake in skeletal muscle may over stimulate cells of the arterial wall and accelerate atherosclerotic process [20]. In recent years, this concept has been expanded to include the idea that not only different cell types, but also different metabolic pathways within the same cell, may differ in their responsiveness to insulin [21].

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2.1. Dysfunctional Energy Storage and Obesity Some investigators regard insulin resistance as a mediating factor in metabolic syndrome, but not as the primary cause [22]. Dysfunctional energy storage seems to be the fundamental issue, being insulin resistance the relevant factor leading to abnormalities in the processing and storage of fatty acids and triglyceride, molecules that account for most of the body’s energy utilization and storage. In most patients, the key abnormality is simply the presence of too much triglyceride, or body fat (i.e obesity). The purpose of adipose tissue throughout the body is energy storage: taking in food calories during and after meals, storing the calories as triglyceride, and then releasing calories in the form of fatty acids when energy is needed [23]. It is safest for the body to store triglyceride in small peripheral adipocytes. If the capacity of these adipocytes to store triglyceride is exceeded, triglyceride accumulates in hepatocytes, skeletal myocytes, and visceral adipocytes. The abnormal triglyceride accumulation may lead to the development of hepatic and muscular resistance to insulin. This is referred to as the “overflow hypothesis” [24]. Visceral adiposity can be measured by waist circumference, waist-hip ratio, or radiographic scans, and it correlates well with insulin resistance and other features of metabolic syndrome. Excess triglyceride in myocytes and in abnormally large peripheral adipocytes appears to engender insulin resistance in these cells [25]. Triglyceride in hepatocytes is recognized as fatty liver and may drive the formation and secretion of excessive VLDL. This theory does not postulate an exclusive role for visceral adiposity, because abnormal peripheral fat cells and triglyceride-laden muscle cells participate in the dysfunctional state [26]. Body mass index and waist circumference tend to load equally in factor analysis, if gender is taken into account. Body mass index may be a sufficient measure for many studies, particularly retrospective analyses of prior data sets. Lipodystrophy syndromes offer striking examples of the effects of the inability to store triglyceride in the physiologically preferred small peripheral adipocytes [27, 28]. The mutations underlying many cases of lipodystrophy are known. These disorders can present in childhood or in adult life with dramatic loss of subcutaneous fat below the shoulder girdle. These patients can develop severe hypertriglyceridemia, insulin resistance, fatty liver, and eventually diabetes mellitus [29]. Patients infected with the human immunodeficiency virus, especially those treated with protease inhibitors; also develop partial lipodystrophy with similarly exaggerated features of metabolic syndrome [30]. Despite their loss of most subcutaneous fat, these patients develop abdominal obesity and fat pads around the base of the neck. In addition to visceral fat, liver, and muscle, excess triglyceride can accumulate in abnormally large peripheral adipocytes. Cross-sectional studies indicate that large subcutaneous adipocyte size is associated with insulin resistance [31, 32]. In a study of Pima Indians, subcutaneous abdominal adipocyte size strongly correlated with risk for developing type 2 diabetes mellitus. This effect of adipocyte size on the risk of developing diabetes was independent of and additive to the effect of insulin resistance [33]. Danforth et al. [27] have proposed that this correlation of adipocyte size and type 2 diabetes mellitus suggests a difficulty in differentiating new adipocytes. A failure of adipocyte differentiation limits the pool of adipocytes available for energy storage, and excess triglyceride overflows to other sites, leading to insulin resistance [34].

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Clinical studies in morbidly obese patients showed that the reduction in body mass index 45 to 35 with surgery is associated with a normalization of insulin sensitivity [35, 36]. When the fat content of muscle was examined in these individuals, it had been reduced to zero, demonstrating that intramyocellular fat content is an important determinant of insulin sensitivity. Fat is found in muscle and liver when it overflows from overwhelmed adipocytes; once fat moves back from muscle and liver to adipocytes, it seems to be stored safely without causing metabolic derangement [37]. Liver along with adipose tissue participate in maintaining glucose and lipid homeostasis through the secretion of various humoral factors and/or neural networks [38, 39, 40]. Various studies have validated the presence of molecular signatures typical of the liver and adipose tissue in mouse models of obesity [41] and in mice fed with a high-fat diet (HFD) [42]. It is believed that perturbations in these “intertissue communications” may be involved in the development of insulin resistance, obesity, and other features of metabolic syndrome [43]. However, it remains unclear which factors alter the communication among tissues and impair the ability of tissues to adapt to changing metabolic states.

2.2. Oxidative Stress and Insulin Resistance Reactive oxygen species production is one of many factors that have been suggested to play a role in the development of insulin resistance, based on the following evidence: i) high doses of hydrogen peroxide [8] and reagents that accumulate ROS [44] can induce insulin resistance in adipocytes, ii) increased markers of oxidative stress were observed in obese humans [45] and rodents [46]. Nevertheless, it remains unclear, whether increased ROS production causes insulin resistance in vivo. It has been demonstrated that the up-regulation of genes responsible for ROS production occurs in both the liver and adipose tissue before the onset of insulin resistance and obesity in mice fed an HFD [47]. It is striking that increased ROS production precedes the elevated levels of TNF-α and FFAs in the plasma and liver in diabetic patients [48]. Reactive oxygen species triggers the development of insulin resistance resulting in abdominal obesity, thereby raising the levels of TNF-α and FFAs. In summary, the HFD induces oxidative stress, potentially through the upregulated expression of genes for ROS production and down-regulation of antioxidant genes, in the liver and adipose tissue [49]. In addition, these changes occur before the onset of insulin resistance and obesity. Sources of ROS induced by an HFD may differ between the liver and adipose tissue. These findings suggest that ROS production may be the initial event triggering HFD-induced insulin resistance and therefore may be an attractive therapeutic target for preventing insulin resistance and obesity caused by an HFD [50].

2.3. Oxidative Alterations, Visceral Obesity and Liver Steatosis A number of clinical studies have reported the importance of visceral fat accumulation in the development of metabolic disorders, including reduced glucose tolerance, hyperlipidemia and cardiovascular diseases [51].

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Visceral fat accumulation causes dysregulation of adipocyte functions, including oversecretion of leptin and TNF-α, plus a diminished secretion of adiponectin. This results in the development of a variety of metabolic and circulatory disorders, including quantitative and qualitative changes in serum lipids and lipoproteins such as small dense LDL [52]. Visceral adiposity represents an independent determinant of all the metabolic syndrome components. In humans, mesenteric fat has been independently associated with body mass index and metabolic risk factors better as compared with measured waist circumference [53, 54]. Abdominal adiposity is also crucial as a source of free fatty acids and inflammatory factors. Indeed, the the International Diabetes Federation gives it a central role in the diagnosis of the metabolic syndrome [55]. Visceral obesity represents per se a low permanent systemic inflammation, as reflected by elevated serum markers, such as C-reactive protein and TNF-α [56]. Genetic manipulation and overnutrition studies have convincingly shown that insulin resistance is regulated by cytokines and mediators released from mesenteric adipocytes [57]. It is generally accepted that the sequence of events leading to hepatocyte fatty degeneration begins with insulin resistance, which precedes fat accumulation [58]. Excess intracellular fatty acids, oxidative stress, energy depletion and mitochondrial dysfunction then cause cellular injury [59, 60]. NASH, the inflammatory form of NAFLD, is thus viewed as the result of “two hits,” in which the first hit is fat accumulation [61]. Lipid retention within hepatocytes triggers oxidative stress (the “second hit”) generating ROS at different intracellular levels and cytokine release. In particular, the alteration of intracellular fatty acid trafficking and mitochondrial β-oxidation, consequent to differential expressions of perilipin and adipophilin [62] and hepatic refractoriness to adipokines [63], contributes to the impairment of hepatic lipid turnover and leads to lipid accumulation. Lipid accumulation and insulin resistance activate different sources of ROS: (i) the cytochrome P450 2E1, which generates ROS during the metabolism of endogenous ketones and dietary constituents [64, 65]; (ii) mitochondria, which continuously generate ROS, being damaged them themselves if the production of ROS is increased [66, 67]; and (iii) peroxisomes, which generate H2O2 and are activated when mitochondrial β-oxidation is saturated or impaired [68]. Enhanced hepatic lipid peroxidation causes changes in physical and chemical membrane properties, with fluidity and permeability alteration [69] affecting signal transduction and ion exchange properties [70]. Changes in lipid composition and characteristics induce membrane remodeling [71]. Most lipid peroxides are volatile molecules that may reach sites distant from those of generation and cause damages and fibroblastic cell activation in the presence of inflammation [72]. In this respect, it has been observed that subjects with NASH show high hepatic and systemic levels of lipid peroxidation products. This phenomenon is associated with an increased risk for cardiovascular disease [73]. Recently, impaired serum redox balance with decreased antioxidant capacity and increased lipid peroxidation has been observed in patients with fatty liver, visceral obesity and metabolic syndrome. In the study by Pou et al. [74], the amount of visceral fat and systemic oxidative alterations were significantly related, thus indicating that excess visceral fat is an important and independent determining factor of the observed serum oxidative changes. Moreover, in these patients, the presence of the metabolic syndrome was predicted from a linear combination of variables, including liver steatosis, visceral fat and serum

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oxidative changes. The participation of the liver both as a damaged organ and a contributory source for systemic oxidative alterations in patients with the metabolic syndrome and visceral adiposity is therefore unequivocally suggested [75]. This role is further supported by the coexistence of fatty liver with blood hypertension and metabolic syndrome in non obese patients [76] and by the observation that NAFLD is associated with the metabolic syndrome to a higher extent than excess adipose tissue in obese subjects [77]. Another clinically relevant aspect is the increased vulnerability of fatty livers toward stress events [78] especially as they occur in transplantation surgery. These mainly depend on the fact that hepatic steatosis sensitizes hepatocytes to injury and inflammation through enhanced fatty acid synthase expression and increased fatty acid synthase-mediated apoptosis [79]. Another potentially damaging factor in NAFLD is intestinal bacteria. The contribution of small bowel bacteria overgrowth to liver inflammatory processes may in fact be realized through an increased intestinal permeability that allows entry of gut-derived toxins with consequent portal inflammation, Kupffer cell activation and liver injury [80, 81]. In this point, NAFLD patients sowed elevated plasma levels of LPS-binding protein (LBP), a biomarker of endotoxemia, and they are further increased in patients with NASH. This increase is related to a rise in TNF-alpha gene expression in the hepatic tissue which supports a role for endotoxemia in the development of steatohepatitis in obese patients [82]. Indeed, ROS are generated in the liver by prooxidant inflammatory pathways that are initiated by gutderived endotoxin [83, 84]. Excess endotoxin can reach the liver through the portal circulation as a result of a higher concentration of endotoxin in the gut or through increased absorption of endotoxin from the gut, i.e.gut leakiness. Wigg et al. [85] compared a group of 22 healthy controls with a group of 23 patients with biopsy-proven NAFLD for the prevalence of small intestinal bacterial overgrowth, increased intestinal permeability and serum endotoxin levels. They found a higher prevalence of small intestinal bacterial overgrowth (assessed by C14-D-xylose breath test) in patients with NAFLD, but found no difference in intestinal permeability (as measured by a lactulose– rhamnose sugar test) or endotoxaemia. Their finding of normal serum endotoxin in the systemic circulation does not exclude endotoxaemia in the portal circulation. They concluded that patients with NAFLD do not have a leaky gut, but bacterial overgrowth may contribute to an endotoxin-initiated hepatic necroinflammatory cascade. However, they did not distinguish between simple steatosis and steatohepatitis. Furthermore, they only studied small bowel permeability. Indeed, loss of colonic barrier integrity in patients with NASH could have a more deleterious effect than loss of permeability of the small bowel, which has relatively low levels of luminal bacteria. Finally, gut leakiness could still be an important pathogenic factor in patients with NASH and ‘normal’ intestinal permeability because these patients may have increased susceptibility to gut leakiness when gut barrier integrity is challenged and results in the intermittent gut leakiness and endotoxaemia necessary to initiate a hepatic necroinflammatory cascade and liver cell injury. In according to this, it have been demonstrated [86] that NASH obese subjects showed a susceptibility to gut leakiness, rather than overt gut leakiness, in with. This susceptibility to leakiness may be the cause of the endotoxaemia and may explain why only a subgroup of patients with NAFLD progresses to steatohepatitis and advanced fibrosis. This finding may

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also help us to find the contributing factors in the pathogenesis of NASH that act disruption of colonic barrier integrity, factors such as NSAIDs, which in turn can lead to endotoxaemia and provide the ‘second hit’ for development of NASH. Based on our results, it is reasonable to recommend to those patients with altered fatty acid metabolism and metabolic syndrome (obesity, diabetes, insulin resistance) and who are more susceptible to oxidative stress to avoid agents that increase permeability such as NSAIDs and alcohol. Larger, interventional or longitudinal prospective studies are needed to assess directly the contribution of susceptibility to gut leakiness in the course of NAFLD.

2.4. Endothelial Oxidative Dysfunction and Blood Hypertension The participation of arterial hypertension in the generation of systemic oxidative stress associated with the metabolic syndrome is suggested by a number of observations on the role of insulin resistance and the sympathoadrenal system [87], NO metabolism changes and the low circulating levels of vitamin C in patients with high-grade hypertension [88] and the improvement of systemic oxidative stress with antihypertensive treatment [89, 90] These considerations have an even higher impact when associated with endothelium activation and dysfunction as characterized by increased levels of circulating oxidized LDL, intercellular and vascular adhesion molecules and C-reactive protein [91] and with the evidence that vascular complications are also associated with oxidative stress events. In particular, small dense LDL particles are able to filtrate through the endothelium of blood vessels. Oxidized LDL activates endothelial cells with the promotion of an immune response leading to the formation of lipid-laden macrophages. Also, in response to inflammatory stimuli, endothelial cells produce adhesion molecules that will further facilitate macrophage migration from the blood into the intimae, thus generating an endothelial damage. These events are favored by insulin resistance and obesity [92]. Other factors have a role in the modulation of these pathophysiologic mechanisms. Among them, vitamin E is a fat-soluble vitamin that is sequestered in the hydrophobic interior of membranes where it acts as an antioxidant, quenching lipid peroxidation. Under normal conditions, the reduced state of LDL is maintained by vitamin E [93, 94], which also acts by regulating inflammatory reactions and metabolic pathways, including platelet aggregation [95]. In combination with tocopherols, vitamin C counters free radicals and regulates vitamin E metabolism by recycling oxidized tocopherols. The synergic action of these two vitamins is also modulated by the intervention of glutathione, which maintains vitamin C in the reduced form [96]. Relatively new and interesting pathways of oxidative stress-induced vascular damages include enzymes such as Nox and homocysteine [97, 98]. Membrane-bound Nox are major sources of ROS in preatherosclerotic conditions and have been found in human peripheral and coronary arteries [99]. A direct spatial relationship between Nox-generated ROS and LDL oxidation was demonstrated in carotid plaques and in lesions associated with unstable angina [100, 101]. By increasing oxidative stress, activation of Nox in vascular cells has been reported to be an important mechanism in the pathogenesis of hypertension and atherosclerosis [102] . Angiotensin II is one of the most potent stimuli activating vascular Nox. This property clearly links ROS production with activation of the renin–angiotensin

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system in hypertension [103]. As a consequence, drugs acting on the renin–angiotensin system reduce Nox activity, thus rendering this enzyme a specific drug target (figure 7-1).

Figure 7-1. ROS generation and consequent oxidative stress as a “second-level” event causing metabolic syndrome-associated. FFA: Free fatty acids; NOX: NADPH oxidase; LDL: Low-density lipoprotein; AOX: Antioxidant defenses; NO: nitric oxide.

Disorders of the folate-dependent methionine metabolism have been described in experimental models and human conditions associated with a high cardiovascular risk [104]. These metabolic abnormalities result in high levels of homocysteine, a molecule belonging to the group of thiols. Differently from glutathione and cysteine, which exert protective effects against ROS, homocysteine is considered to be a “bad thiol” because of its association with a variety of chronic disease conditions [105]. Homocysteine is formed in the transsulfuration and remethylation pathways that convert homocysteine to methionine with folate and betaine

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intervention. As a consequence of the impairment of the methionine metabolism, the increased level of circulating homocysteine has been associated with endothelial dysfunction, both directly and via NO interaction [106]. Although it is known that homocysteine can be toxic per se by acting as an N-methyl-D-aspartate agonist, thus decreasing the availability of NO and impairing arterial vasodilation capacity [107], the specific molecular mechanisms of damage involved are still unclear. Three mechanisms have been proposed, namely, oxidative stress, endoplasmic reticulum stress and activation of pro-inflammatory factors [108]. Hyperhomocysteinemia has been associated with oxidative stress in liver steatosis, a hypothesis favored by recent observations [109, 110]. An additional mechanism that may be involved in homocysteine-mediated vascular alteration in patients with the metabolic syndrome is the indirect connection between high homocysteine levels and low nitrosothiol levels. At this concern, it is known that circulating nitrosothiols act as free NO donors for vascular tone modulation [111]. In addition, by representing a storage form of thiols and glutathione in particular, extracellular nitrosothiols exert antioxidant functions and favor removal of toxic products [112, 113], contributing to the role of the extracellular microenvironment in the regulation of the redox status and function of cell surface proteins [114]. The above-reported interference of homocysteine with circulating NO availability could explain, at least in part, the low levels of nitrosothiols and glutathione found in conditions associated with hyperhomocysteinemia [115]. Therefore, the equilibrium between “good” and “bad” thiols may determine outcomes in studies of tissue degeneration and inflammation (i.e., NASH and the metabolic syndrome).

2.5. Experimental Studies of the Metabolic Syndrome Animal models can be helpful in further understanding the potential pathophysiology of the metabolic syndrome. Murine models in particular have become quite useful tools in recent years because the entire mouse genome is now sequenced, and a large number of transgenic and knockout models are readily available. There are a number of limitations with these models, however, that must be considered. Rodent lipid physiology, for example, is significantly different compared with humans. Rodents carry most of their cholesterol in HDL, not LDL; thus, a low level of HDL is an unusual finding. Blood pressure is usually not measured in these models, again limiting the use of the “human” clinical definition of the metabolic syndrome. Nevertheless, there remains much to be learned from animal models that may be applicable to mechanisms of the metabolic syndrome in humans. 2.5.1. Mouse Models Various murine models exhibited many of the components of the metabolic syndrome, i.e., leptin-deficient ob/ob and leptin-resistant db/db mice [116]. More recently, when ob/ob mice were crossed with the LDL-receptor-deficient mouse, the features of the metabolic syndrome including obesity, dyslipidemia, hypertension, insulin resistance and impaired glucose tolerance, and/or diabetes plus hypercholesterolemia resulted in more oxidative stress and atherosclerosis [117, 118]. A number of less-well known polygenic mouse models have a mixture of components of the MetS and its associated diseases. It is worth noting that mice

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with different genetic backgrounds have a variable propensity to develop the MetS in response to changes in diet composition [119, 120]. For instance, when C57Bl/6 (B6) and 129S6/SvEvTac [121] mice were placed on a low-fat or high-fat diet for 18 wk, the 129 strain developed features of the metabolic syndrome, notably obesity, hyperinsulinemia, and glucose intolerance only on the high-fat diet, whereas the B6 strain developed these features on both diets [122]. The Jackson Laboratory has carried out a comprehensive assessment of genetic susceptibility to the metabolic syndrome in inbred mice when challenged with a high-fat, high-cholesterol diet [123]. A standard protocol was set up to evaluate female and male mice from 43 inbred strains for 10 traits including all the major criteria of metabolic syndrome while mice consumed the diet for 18 wk. A few strains of mice developed a phenotype with a plethora of metabolic abnormalities remarkably similar to the human metabolic syndrome (strains CAST/EiJ, CBA/J, and MSM/Ms). Other strains had a more limited phenotype, i.e., severe obesity (AKR/J and KK/HIJ) vs. protection from obesity (WSB/EiJ); severe dyslipidemia (MOLF/EiJ) vs. no dyslipidemia (CZECHII/EiJ for males and D2 for females); and severe insulin resistance (KK/HIJ) vs. being spared from insulin resistance (A/J). Overall, the discrepant phenotypes within the same environmental exposures may prove useful in dissecting the genetic and related molecular mechanisms underlying the metabolic syndrome and its components [124]. 2.5.2. Rat Models A number of models of the metabolic syndrome have been identified in rats. The Zucker fatty rat was among the first identified [125]. Subsequently, a number of studies have been published to examine the impact of diet on the phenotypic development of the metabolic syndrome [126-128]. Wistar Ottawa Karlsburg W rats (WOKW) develop all components of this syndrome. Genetic analysis of this rat model has identified potential major quantitative trait loci (QTL) for glucose metabolism on chromosome 3, dyslipidemia on chromosomes 4 and 17, and obesity on chromosomes 1 and 5 [129]. Moreover, the severe insulin resistance predominant in epididymal adipose tissue of these rats was associated with a 10-fold decrease in adipocyte adiponectin gene expression and decreased peroxisome proliferator-activated receptor gene expression, but increased FOXO1 gene expression compared with control rats [130]. Moreover, the metabolic in WOKW rats was associated with impaired coronary vasodilatation due to altered adrenoceptor sensitivity [131]. Another example in rats is the corpulent (JCR: LA-cp) rat that like db/db in mice is a homozygous mutation in the leptin receptor [132]. These rats are obese, insulin resistant, and hypertriglyceridemic. JCR:LA-cp rats, however, are prone to atherosclerosis [133, 134] and also appear to be a good model to study the contribution of postprandial lipemia to the atherosclerotic process. In these rats, lymphatic chylomicron apoB48, fasting and postprandial plasma apo B48 area under the curve are all elevated [135]. The Prague hereditary hypertriglyceridemic (hHTG) rat was developed as a model of hypertriglyceridemia. Although these rats are not obese, they are hypertensive, insulin resistant, and glucose intolerant [136]. Using F2 hybrids, several QTL have been identified for hypertension and hypertriglyceridemia [137]. Another model of the metabolic syndrome in rats that includes hypertension is the Lyon hypertensive rat (LH). These rats also have

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obesity, dyslipidemia, and an increased insulin/glucose ratio. This rat strain has been used to identify linkage of body weight, blood pressure, and renal, metabolic, and endocrine phenotypes [138]. This is a rennin-dependent model of hypertension in which low-dose (nonantihypertensive) ACE inhibitor therapy affords significant and durable renal protection. A total genome scan in the offspring of an F2 intercross between the hypertensive and normotensive Lyon strains has identified a series of QTL for the metabolic syndrome, body weight, blood pressure, lipid metabolism, and renal function [139]. Other hypertensive rat models of the metabolic syndrome include SHR/NDmccp(cp/cp) [140] and SHROB (spontaneously hypertensive, obese rat) [141]. 2.5.3. Other Animal Models Of interest to pet owners and veterinarians alike is the fact that obesity in dogs and cats has increased in recent years [142], and dogs in particular are models of the metabolic syndrome. The canine obesity model closely recapitulates the relationship between human visceral adiposity and insulin resistance. The work of Bergman et al., [143] supports the portal theory of insulin resistance, in which FFA from visceral adipose tissue directly enter the liver and unfavorably modify insulin action. Sympathetic nervous system hyperactivity in this model of obesity may also contribute to excessive free fatty acids (FFA) release, hypertension, and insulin resistance. As noted previously, a nocturnal increase in plasma FFA levels may account for both insulin resistance and compensatory hyperinsulinemia. Obesity is common in cats and is a risk factor for diabetes. The prevalence of diabetes has increased concomitantly with the increase in obesity, and diabetes is now seen in approximately 0.5–1% of cats [144]. Cats develop a form of diabetes similarly to type 2 diabetes in humans, characterized by islet amyloid accumulation and loss of β-cell mass [145]. From more recent studies in felines, it appears that glucose metabolism in cats is similar to that in humans; however, lipid metabolism is quite different [146].

3. Role of Antioxidants in Attenuation of Metabolic Syndrome Progression 3.1. Experimental Studies 3.1.1. Antioxidant Vitamins There is direct evidence that micronutrients have a beneficial effect on insulin sensitivity and some components of the antioxidant defense system in an animal model of insulin resistance [147]. In this point, the beneficial effects of antioxidant vitamins supplementation are attributed to their ability to scavenge free radicals, control nitric oxide synthesis or release, inhibit reactive oxygen species generation and upregulate antioxidant enzyme activities that metabolize these molecules [148]. Low levels of vitamin C, a potent dietary antioxidant molecule, have been associated not only with obesity [149] but also with a variety of conditions including hypertension, gallbladder disease, stroke, some cancers and atherosclerosis [150]. Moreover, vitamin C administration ameliorates hyperglycemia and glycosylation in diabetic-obese rodents [151] and inhibits the activation of inflammatory

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response mediated by nuclear factor-kappa B [152]. In addition, to the health effects of ascorbic acid as antioxidant, this vitamin could be involved in obesity-related mechanisms, for example, regulation of behavioural activity [153], lipolysis [154] and glucocorticoid release from the adrenal glands [155]. In hypertensive rats long term Vit C administration significantly reduced systolic blodd pressure and simultaneously reduced oxidative stress mediated by NAD(P)H oxidase activation [156]. Vitamin C has beneficial effects not only on blood pressure but also on endothelial function in hypertensive and diabetic patients [157]. Vitamin C is a soluble compound and it prevents protein and lipid oxidation in the extracellular environment [158]. In vivo studies confirmed that vitamin C administration improves arterial vasodilatation by increasing NO production [159]. Demonstration of free radical damage and its prevention by vitamin E in vivo have lagged because of a lack of sensitive analytical techniques. This, however, has recently changed; quantification of F2-isoprostanes, isomers of prostaglandin F2, has been suggested by a number of investigators as a reliable index of in vivo free radical generation and oxidative lipid damage. F2-isoprostanes are formed in membranes from arachidonylcontaining lipids by cyclooxygenase enzymes, as well as during free radical-catalyzed lipid peroxidation [160]. In studies using experimental animals, F2-isoprostanes increased in plasma and tissues as a result of vitamin E deficiency [161]. Furthermore, in an animal atherosclerosis model (the apoE-deficient mouse), vitamin E supplementation not only suppressed F2-isoprostane production but also decreased atherosclerotic lesion formation [162]. In other animals models in which that estrogen deficiency or ovariectomy results in a reduction of sexual steroids and increased prevalence of cardiovascular diseases, it was assessed the benefits of antioxidant vitamins (E and C) for the protection against cardiovascular disease and oxidative stress. The adjunct antioxidant treatment potentiated the hormone replace treatment and showed a significant correction of homocysteine and GSH levels [163]. Further studies are warranted to elucidate the beneficial role of antioxidant treatment of cardiovascular protection of estrogen deficiency models, relevant to elucidate clinical complications that present of menopause women. 3.1.2. Flavonoids Many observational and experimental studies have considered that caloric restriction may be associated with life prolongation [164]. possibly through an improvement of the cell redox balance [165]. Also, increased generation of mitochondrial ROS and oxidative damages seem to be differently induced by nutritional perturbation and state [166]. In animal experiments hypocaloric diet and antioxidant supplementation were associated with improvement of some tissue functions and redox states that, conversely, were oxidatively depressed in aged control animals [167]. A key event associated with diet restriction is the activation of a class of genes belonging to the Sirt family, which is involved in cell maturation and apoptotic processes. Recently, Howitz et al. [168] showed that resveratrol, an antioxidant poliphenol of red wine, was able to activate these genes by mimicking the effect of diet restriction. Successively, Baur et al. [169] showed that high dose resveratrol was able to contrast the development of cardiovascular diseases and diabetes in mice that underwent a hyperlipidic diet, suggesting a role for oxidative stress in systemic inflammation and damages in conditions simulating the

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metabolic syndrome. In this point, grape extracts enriched in different polyphenolic families have been utilized to prevent reactive oxygen species (ROS) production, although having differential effects on various features of metabolic syndrome when administered to the fructose (60%)-fed rat (a model of metabolic syndrome) [170]. The effect of pure polyphenolic molecules (catechin, resveratrol, delphinidin, and gallic acid) prevented insulin resistance, the elevation of blood pressure and cardiac ROS overproduction and NADPH overexpression. Indeed, fructose feeding is associated with cardiac fibrosis (accumulation of collagen I) and expression of osteopontin, a factor induced by ROS and a collagen I expression inducer. In this model, collagen I and osteopontin expressions could be prevented by the administration of polyphenolic molecules [171]. The potential use of polyphenols in the prevention of cardiac complications associated to metabolic syndrome should be further explored. 3.1.3. Other Antioxidants Systemic oxidative stress and nitrative stress as well a inflammation increase with the development of metabolic syndrome-like components in SHR/ cp rats, which display abdominal obesity, hypertension, hyperglycemia, insulin-resistance, and hyperlipidemia [172]. Long-term CoQ10 administration can prevent increased oxidative and nitrative stress [173], as indicated by higher levels of Ox-LDL and 8- OHdG in the serum and of 3nitrotyrosine in serum proteins, respectively, and the increased inflammation with activation of myeloperoxidase, as indicated by higher serum levels of C-reactive protein and 3chlorotyrosine in the SHR/ cp rats displaying metabolic-like components. In addition, the elevated serum insulin levels and high blood pressure were suppressed by CoQ10 intake for 10 weeks. In diabetic rats, CoQ10 treatment also reduced lipid peroxidation and increased antioxidant parameters like superoxide dismutase, catalase, and glutathione in the liver homogenates of diabetic rats. CoQ10 also lowered the elevated blood pressure in diabetic rats, explained to mechanism based on induction of antioxidant defense system [174]. CoQ10 prevents vascular endothelial dysfunction seem to be linked to its hypotensive effect in SHR/ cp rats. Furthermore, insulin resistance and the consequent hyperinsulinemia, important components of metabolic syndrome, are associated with endothelial dysfunction, probably due to increasing oxidative stress [175]. The physiological properties of insulin that cause enhancement of renal sodium reabsorption and stimulate sympathetic nervous system activity are believed to play a major role in the development of hypertension [176], although the underlying mechanisms in the setting of insulin resistance remain obscure [177]. Therefore, the hypotensive effect of CoQ10 observed in SHR/cp rats may be associated with its alleviation of hyperinsulinemia together with endothelial dysfunction. These findings suggest that the antioxidant properties of CoQ10 can be effective for ameliorating cardiovascular risk in metabolic syndrome.

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3.2. Clinical Studies 3.2.1. Antioxidants Vitamins Both vitamins, E and C, appear to be important for the prevention of cardiovascular events. In fact, consumption of vitamin E has been associated with a lower risk for coronary heart disease [178] and with reduced LDL oxidation [179]. Also, the connection between serum concentrations of vitamin E and lipid peroxidation products in relation to cholesterol level and abdominal obesity has been recently studied in patients with the metabolic syndrome. An inverse relation between the serum cholesterol-adjusted vitamin E concentrations and the grade of hepatic steatosis and a linear relation between the extent of visceral fat and the lipid peroxide/ cholesterol ratio have been observed [61]. In other studies, supplementation of vitamin E was able to prevent the onset of type 2 diabetes [180] and improve NAFLD in obese children [181]. All of the studies discussed were carried out with alpha-tocopherol supplements. Alpha-tocopherol decreases plasma gamma-tocopherol concentrations, as a result of the function of the hepatic alpha-tocopherol transfer protein, which preferentially incorporates alpha-tocopherol into the plasma [182], as well as increasing gamma-tocopherol metabolism. This observation has often been suggested as an explanation for the null results observed with alpha-tocopherol supplementation in the majority of prospective clinical trials, especially since gamma-tocopherol concentrations are inversely associated with increased morbidity and mortality due to cardiovascular disease [183]. Recently studies showed [184, 185]. that in metabolic syndrome-subjects the combination of alpha-tocopherol y gamma-tocopherol therapy results in significant reductions in C-reactive protein, urinary nitrotyrosine, and lipid peroxides. Future studies will be directed at examining mechanisms for these changes and testing the effect of combined supplementation on cardiovascular events in high risk populations such as chronic kidney disease and metabolic syndrome. Concerning the specific role of vitamin C in oxidative stress-associated arterial hypertension, mounting evidence suggest the importance of this vitamin in regulating endothelial function and vasodilation. In fact, vitamin C is known to improve elastic artery [186], by contrasting endothelial cell oxidation and by stimulating both endotheliumdependent and endothelium-independent arterial vasodilation [187]. In addition, vitamin C administration was able to restore endothelium-dependent vasodilation in hyperglycemic patients [188]. 3.2.2. Flavonoids The Mediterranean diet contains a high rate of olive oil, fish, vegetable and low consumption of alcohol, thus spreading a wide antioxidant capacity. The Mediterranean diet has also been associated with a reduced incidence of blood hypertension, suggesting that a diet regimen well balanced in carbohydrates and fats could be indicated to correct metabolic abnormalities in metabolic syndrome patients. In a recent controlled crossover trial [189], lower plasma oxidized LDL and lipid peroxide levels and higher glutathione peroxidase activity were observed after an olive oil intervention, suggesting that consumption of olive oil, rich in phenolic antioxidant compounds, could provide beneficial effects in patients with cardiovascular risk factors. In this respect, it is also known that dietary fats can accomplish

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regulation of hepatic lipid metabolism through modification of gene transcription [190]. This is achieved by long-chain polyunsaturated fatty acids that are able to direct (i) fatty acids away from triglyceride storage by enhancing their oxidation and; (ii) glucose away from fatty acid synthesis by increasing its flux to glycogen [191]. Increased consumption of fruits and vegetables has also been shown to be associated with a reduced risk for stroke in most epidemiological studies [192, 193]. In a recent metaanalysis of prospective cohort studies, He et al. [194] demonstrated that intake of fruits and vegetables higher than the average of three servings per day was associated with a lower risk for stroke, thus providing strong support for the use of antioxidant vitamin-rich food in the diet of patients with cardiovascular risk factors. However, experimental and human studies of the addition of antioxidants to diets and other treatments in patients with NASH and metabolic syndrome yielded controversial results. In particular, although a vitamin Edeficient diet elevated the lipid peroxidation levels in the rat liver, both ubiquinol and glutathione seem to protect mitochondria from lipid peroxidation more than vitamin E [195]. In humans, whereas addition of vitamin E to ursodeoxycholate in the treatment of NASH patients improved laboratory test and hepatic histology findings in a small number of metabolic syndrome patients [196], a combined vitamin E and vitamin C treatment did not improve necro-inflammatory activity or alanine aminotransferase and was not superior to weight loss in reducing biochemical indexes in two different studies of NASH patients [197]. In this point, is relevant mentioned some studies that demonstrate scavenger properties that polyphenolic compounds in chronic consumtion in patients with NASH, however these benefits are not expressed in functional parameters [198]. 3.2.3. Other Antioxidants Another natural food compound with protective properties is betaine. Betaine is distributed widely in plants (wheat germ, bran and spinach), and rich dietary sources include seafood, especially marine invertebrates [199]. The principal physiologic role of betaine is the methyl donor (transmethylation) in the methionine cycle. Inadequate dietary intake of betaine leads to disturbed hepatic methionine metabolism resulting in elevated plasma homocysteine concentrations and to inadequate hepatic fat metabolism leading to steatosis and subsequent increased serum lipid levels. These metabolic alterations may contribute to coronary, cerebral, hepatic and vascular diseases. Betaine has been shown to protect internal organs, improve vascular risk factors and enhance performance [200]. Coenzyme Q10 (CoQ10) is an endogenously synthesized compound that acts as an electron carrier in the mitochondrial respiratory chain [201]. In addition to its unique role in mitochondria, CoQ10 functions as an antioxidant, scavenging free radicals and inhibiting lipid peroxidation [202]. Recent studies have provided evidence of the potential value of CoQ10 in prophylaxis and therapy of various disorders related to oxidative stress. There is promising evidence of the beneficial effect of CoQ10 in hypertension and heart failure [203]. It has been reported that CoQ10 concentrations and redox status are associated with components of metabolic syndrome [204]. The administration of CoQ10 notably suppresses oxidative and nitrative stress, inflammation, hypertension, and hyperinsulinemia [205]. These findings suggest that the antioxidant properties of CoQ10 can be effective for ameliorating

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cardiovascular risk in metabolic syndrome. Table 1 shows a summary of some clinical trials that support interventions with antioxidant in metabolic syndrome. Finally, natural elements appear to have a role in the regulation of serum glycemia and associated metabolic dysfunctions. In particular, some observations have suggested that excess intake of refined carbohydrates is associated with decreased levels of serum chromium [206] and that this element has potential benefits on hyperglycemia, diabetes and elevated serum lipids [207]. It has been suggested that chromium explicates its action by improving some insulin effects, including the glucose transport within mitochondria, and improving the energetic demand. Table 7-1. Clinical trial that support the antioxidant interventions in metabolic syndrome Compound

Mechanism

Effects in Human

Vitamin C

↓ ROS production

Improve endothelial function Schneider et al., 2005 Moreau et al., 2005

Vitamin E

Scavenger ROS

↓ Endothelial oxidation May & Qu, 2005

Scavenger ROS

↓ LDL oxidation Lapointe et al., 2006

Antiinflammatory

↓ C-reactive protein Skalicky et al., 2008

Flavonoids

↓ ROS production

↓ LDL oxidation Fito et al., 2004 Feillet-Coudray et al., 2008

Scavenger ROS

↓ Liver steatosis Kaviarasan et al., 2007

Betaine

↓ Homocysteinemia

Improve vascular function ↓ Liver steatosis Craig , 2004

Coenzyme Q10

Scavenger ROS

↓ Hyperinsulinemia ↓ Blood pressure Rosenfeldt et al., 2007 Pepe et al., 2007

ROS: Reactive oxygen species; LDL: Low density lipoprotein.

4. Conclusions and Perspectives The metabolic syndrome is common, and the associated risk burdens of diabetes and cardiovascular disease are a major public health problem. The hypothesis that the main

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constituent parameters of the metabolic syndrome share common pathophysiologic mechanisms of damage provides a new conceptual framework for future research, although clinical trials will be necessary to confirm that the results from animal studies are applicable to humans. Actually is the well-documented beneficial effects of exercise and body weight reduction in the prevention of insulin resistance and in the amelioration of diseases associated with the metabolic syndrome, the paradigm discussed in this review suggests that interrupting intracellular and extracellular ROS overproduction would contribute to normalizing the activation of metabolic pathways leading to the onset of diabetes and its complications and contrast the appearance of endothelial dysfunction leading to cardiovascular complications. This view supports the hypothesis that oxidative stress, mechanistically explaining the perpetuation of insulin resistance and endothelial dysfunction, may contribute to the appearance of cardiovascular complications in patients with the metabolic syndrome. Under conditions of elevated metabolism, many tissue-specific cells are continuously subject to insult from ROS, such as the superoxide radical and H2O2. This is probably a common feature for elements of the metabolic syndrome such as hypertension, hypertriglyceridaemia, diabetes and obesity. Moreover, an increase in ROS production is one of the earliest events in cases of glucose intolerance, and it may be the cause of pancreatic βcell dysfunction as well as hepatic pathologies. Interestingly, β-cells produce ROS in response to increased glucose concentrations, but express relatively low levels of freeradical-detoxifying enzymes. This combination might make β-cells particularly sensitive to oxidative stress. It is also becoming more appreciated that hepatic steatosis and steatohepatitis are closely related to the generation of ROS. Nonetheless, drugs currently approved for use in clinical practice are highly effective for the treatment of modifiable risk factors, and notably hypertension and dyslipidaemia. At present, however, physicians tend to target cardiovascular risk factors in isolation, and as a direct consequence of treating individual risk factors. In order to obtain maximal reductions in cardiovascular disease events and to optimise clinical benefit, therapeutic strategies which target multiple cardiovascular risk factors for the management of global cardiovascular risk should be used. An integrated approach to the control of blood pressure and dyslipidaemia, alongside interventions to improve insulin sensitivity, weight loss, and reduce smoking, may therefore represent an effective therapeutic strategy for the attenuation of atherogenesis and the prevention of cardiovascular disease in high-risk patients. Future research should help further define the potential role of antioxidant supplementation to diet and exercise. Indeed, for many years, interest has focused on strategies that enhance removal of ROS using either antioxidants or drugs that enhance endogenous antioxidant defense. Although those strategies have been effective in experimental models, several trials have shown that they do not reduce cardiovascular events and in some cases have actually worsened the outcome. An intriguing alternative approach to reduce oxidative stress is inhibiting ROS production by blocking enzymes involved in its synthesis. This hypothesis opens testing novel molecules that could interfere with the production of free radicals and may result in reversing, or even retarding, diseases caused by oxidative and inflammatory processes, such as the metabolic syndrome.

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In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter VIII

Diabetes Mellitus Rodrigo Castillo Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile. Supported by FONDECYT, grant 1070948

Abstract Elevation of glycemia in diabetic patients may lead to the autooxidation of glucose, glycation of proteins, and the activation of polyol metabolism. These changes accelerate the generation of reactive oxygen species (ROS) and increase oxidative modification of lipids, DNA, and proteins in various tissues. Thus, oxidative stress occurring in this setting may play an important role in the development of the chronic complications of diabetes, such as nephropathy, neuropathy, and lens cataracts. Langerhans islets are more vulnerable to the occurrence of oxidative stress, since they contain low levels of antioxidant enzyme activities compared to other tissues. High glucose concentrations are known to give rise to a manifestation named glucose toxicity. Major manifestations of glucose toxicity in the pancreatic β-cells are defective insulin gene expression, diminished insulin content, and defective insulin secretion. The link between the clinical complications and oxidative stress-related parameters has been established by the study of advanced glycation end products (AGEs). Among the latter, heterocyclic amines, acrylamide, and AGEs are well-known compounds hypothesized to cause harmful health effects. First, AGEs act directly to induce cross-linking of long-lived proteins, such as collagen, to promote vascular stiffness, thus altering the structure and function of vasculature. Second, AGEs can interact with their receptors to induce intracellular signaling leading to enhanced oxidative stress and elaboration of key proinflammatory and prosclerotic cytokines. Over the last decade, a large number of preclinical studies have been performed, targeting the formation and degradation of AGEs, as well as their interaction with specific receptors. Translational research with humans is now under way to ascertain whether this protection can be provided to patients experiencing inadequate glycemic control.

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1. Introduction Diabetes mellitus is a major cause of morbidity in the western world, and of the most common severe chronic illnesses, affecting over 230 million people worldwide with an estimated global prevalence of 5.1% [1]. The associated complications, mainly coronary disease, poses enormous public health and economic burdens, novel preventive and regenerative therapies have emerged in the past decade with the aim to preserve pancreatic βcell mass and delay the onset of diabetes. This illness is characterized by a chronic metabolic disorder caused by defects in both insulin secretion and action. An elevated rate of basal hepatic glucose production in the presence of hyperinsulinemia is the primary cause of fasting hyperglycemia. In this setting, after a meal, impaired suppression of hepatic glucose production by insulin, and decreased insulin-mediated glucose uptake by muscle, contribute almost equally to postprandial hyperglycemia. The reason for the injury related to hyperglycemia is the formation of advanced glycation end products (AGE) such as glycated proteins, glucose oxidation-derived metabolites, and increased free fatty acids [2]. These effects result in oxidative stress in the mitochondria, as well as in the activation of oxidative and inflammatory signaling pathways. The latter is continued with damage to the insulin-producing cells, resulting in various complications of diabetes. The majority of these complications, including retinopathy, nephropathy, atherosclerosis and subsequent coronary artery disease, cerebral vascular disease, and peripheral artery disease, can be related to microangiopathy or endothelial injury [3]. It is clear that glucose toxicity can result in abnormal fatty acid metabolism, namely autooxidation of glyceraldehyde, which generates hydrogen peroxide and ketoaldehydes. This can lead to chronic oxidative damage. In the presence of reactive metals, hydrogen peroxide could form the hydroxyl radical leading to toxicity. In addition, glucose toxicity results in protein kinase C activation and its downstream effects on transforming growth factor β, vascular endothelium growth factor, endothelin 1, and nuclear factor κB, among others [4]. Therefore, the formation of glycation products and sorbitol is important in the pathogenesis of the complications of diabetes. These same products are involved in the process of aging, resulting in DNA strand breaks and production of reactive dicarbonyls [5]. The most concerning aspect of the disease is the functional impairment in β-cell caused by oxidative stress, resulting in a loss of insulin gene expression in the islet cells. Furthermore, the hyperlipidemia that often accompanies diabetes mellitus can result in fatty acid-mediated oxidative damage and metabolic disturbances in the β-cells [6]. Although the use of antioxidants has been proposed to prevent some of the complications of diabetes [7, 8], this intervention may provide only a partial solution. Nonetheless, it is important to understand the role of oxidative stress in the disease process of diabetes. This chapter is aimed to show the state of the art about the insights that will foster further investigation into the mechanisms by which oxidative stress influences the onset and progression of diabetes. In addition, the rationale suggesting potential therapeutic and preventative measures for this frequent condition, based on the use of antioxidants, will be discussed.

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2. Pathophysiology of Diabetes Mellitus 2.1. Type 1 Diabetes Type 1 diabetes is presented as a chronic immune-mediated disease with a subclinical prodromal period characterized by selective loss of insulin-producing β-cells in the pancreatic islets in genetically susceptible subjects. Autoreactive T cells, both CD4 and CD8, have been implicated as active players in the β-cell destruction. The most important genes contributing to disease susceptibility are located in the HLA class II locus on chromosome 6. In addition, ten other genes or genetic regions have also been associated with this disease [9]. A series of autoantigens have been identified, including insulin, glutamic acid decarboxylase, the protein tyrosine phosphatase-related islet antigen 2, and most recently the zinc transporter Slc30A8 residing in the insulin secretory granule of the β-cell [10]. Nevertheless, only a relatively small proportion, less than 10%, of individuals with HLA-conferred diabetes susceptibility progress to clinical disease. This implies that additional factors are needed to trigger and drive β-cell destruction in genetically predisposed subjects. Clinical type 1 diabetes represents end-stage insulitis, a histologic change in the islets of Langerhans characterized by edema and lymphocyte infiltration, and it has been estimated that at the time of diagnosis only 10–20% of the β-cells are still functioning. It is generally accepted that the destruction of the β-cells is mediated by cellular immune responses. This is supported by the following facts: (i) T cells are present in insulitis; (ii) disease progression is delayed by immunosuppressive drugs directed specifically against T cells; and (iii) circulating autoreactive T cells can be detected in patients at clinical presentation of type 1 diabetes [11]. It has remained open where potentially islet-autoreactive T lymphocytes are activated initially. The activation of T cells requires the presentation of autoantigenic determinants to self-reactive T cells by major histhocompatibility complex (MHC). The MHC is a set of molecules displayed on cell surfaces that are responsible for lymphocyte recognition and antigen presentation. Indeed, the MHC molecules control the immune response through recognition of our own cells, and reacting against foreigner antigens. However, MHC II molecules may not be expressed normally on β-cells in vivo. It has been shown by in vitro experiments that the expression of MHC II molecules can be induced on the surface of the β-cells by the combined effect of interferon-γ and TNF-α [12], making possible the locally activation of naïve autoreactive T cells in the islets. Alternatively, and even more likely, autoantigens are presented to naïve autoreactive T cells by antigen-presenting cells (APC) which primarily express MHC II molecules. It has been postulated that the initial encounter of APC and naïve self-reactive T cells takes places in the pancreatic lymph nodes. The activated T cells are capable of invading the islets, where they become reactivated by encountering cognate β-cell autoantigens and thereby initiating insulitis. It seems that the autoimmune response is antigen-driven in type 1 diabetes. This is supported by the fact that the strongest genetic susceptibility is associated with MHC class II alleles. Recent studies have shown that type 1 diabetes is a proinflammatory state [13, 14]. Schalkwijk et al. [15] reported elevated C-reactive protein (CRP) levels in Type 1 diabetes, compared with controls patients without macrovascular disease. In the EURODIAB study

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[16], levels of CRP, plasma interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were found high in type 1 diabetic subjects and, in a cross-sectional study, correlated with the severity of diabetic vascular disease. In this point, animal models of type 1 diabetes provide strong support to the hypothesis that Toll-like receptor-induced innate signaling pathways are involved in the proinflammatory process leading to autoimmune diabetes [17]. Studies performed in peripheral blood cells and sera from patients with type 1 diabetes indicate that aberrant innate functions might exist in such patients [18], but the relevance of these alterations to the mechanism leading to type 1 diabetes is currently unclear. These observations indicate that any intervention manipulating the autoimmune response would have the highest likelihood of success, if initiated as soon as possible after the appearance of the first signs of β-cell autoimmunity. There may be a critical window of 1 year for immune intervention after the emergence of the first signs of the disease process in type 1 human diabetes.

2.2. Type 2 Diabetes In patients with type 2 diabetes and established fasting hyperglycemia, the rate of basal hepatic glucose production is excessive, despite plasma insulin concentration that are increased two-fold to four-fold [19]. These findings provide unequivocal evidence for hepatic resistance to insulin, and this evidence is substantiated by an impaired ability of insulin to suppress hepatic glucose production. Accelerated gluconeogenesis is the major abnormality responsible for the increased rate of basal hepatic glucose production. The increased rate of basal hepatic glucose production is closely correlated with the increase in fasting plasma glucose level [20]. Muscle tissue in patients with type 2 diabetes is resistant to insulin [21. Defects in insulin receptor function, insulin receptor-signal transduction pathway, glucose transport and phosphorylation, glycogen synthesis, and glucose oxidation contribute to muscle insulin resistance. In response to a meal, the ability of endogenously secreted insulin to augment muscle glucose uptake is markedly impaired [22]. Muscle insulin resistance and impaired suppression of hepatic glucose production contribute almost equally to the excessive postprandial increase in the plasma glucose level [23]. Under diabetic conditions, ROS [24] are produced in various tissues such as nerve cells and vascular cells, and are involved in the development of diabetic complications [25, 26]. Recently, pancreatic β-cells emerged as a target of oxidative stress–mediated tissue damage [27, 28]. Pancreatic β-cells express the high Km glucose transporter GLUT-2 abundantly and thereby display highly efficient glucose uptake when exposed to high glucose concentration. Also, because of the relatively low expression of antioxidant enzymes, such as catalase and glutathione peroxidase [29], pancreatic β-cells may be rather sensitive to ROS attack when they are exposed to oxidative stress. Thus, it is likely that oxidative stress plays a major role in β-cell deterioration in type 2 diabetes. There are several sources of ROS production in cells: the nonenzymatic glycosylation reaction [30, 31], the electron transport chain in mitochondria [32], and the hexosamine pathway [33]. Among these, the glycation reaction seems to have broad pathological

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significance in diabetic complications, because under hyperglycemia the production of various reducing sugars, such as glucose, glucose-6-phosphate, and fructose, increases through glycolysis and the polyol pathway. All of these reducing sugars are known to promote glycation reactions of various proteins. In diabetic animals, glycation is observed extensively in various tissues and organs, and various kinds of glycated proteins. The latter might include glycosylated hemoglobin, albumin, and lens crystalline produced in a nonenzymatical manner through the Maillard reaction. During this reaction, which in turn produces Schiff bases, Amadori product and AGE, ROS are also produced [34]. Also, the electron transport chain in mitochondria is likely to be an important pathway to produce ROS. Indeed, it was suggested that mitochondrial overwork, which causes induction of ROS, is a potential mechanism leading to impaired first-phase of glucose stimulated insulin secretion found in the early stage of diabetes. In addition, ROS are produced also through the hexosamine pathway and that activation of the pathway leads to deterioration of β-cell function by provoking oxidative stress [35] (figure 8-1).

Figure 8-1. Sources of cellular ROS in chronic hyperglycemia TCA, Tricarboxylic acid.

2.3. Advanced Glycation End Products and Oxidative Stress The increasing body of evidence targeting accumulation of AGEs and/or their receptors (RAGE) that mediate the biological actions could potentially confer benefits on diabetesrelated end-organ injury [36]. Advanced glycation end products are a complex group of compounds formed via a non enzymatic reaction between reducing sugars and amine residues on proteins, lipids, or nucleic acids. The major AGEs in vivo appear to be formed from highly reactive intermediate carbonyl groups, known as α-dicarbonyls or oxoaldehydes, including 3-

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deoxyglucosone, glyoxal, and methylglyoxal [37, 38]. Some of the best chemically characterized AGEs in humans include pentosidine and N-carboxymethyl lysine. Apart from endogenously formed products, AGEs can also originate from exogenous sources such as tobacco smoke and diet [39, 40]. Food processing, especially prolonged heating, has an accelerating effect in the generation of glycooxidation and lipid oxidation products, and a significant proportion of ingested AGEs is absorbed with food. Tissue and circulating AGE levels are higher in smokers and in patients on high AGE diets, with concurrent increases in inflammatory markers [41]. Furthermore, there is evidence from animal studies that exposure to high levels of exogenous AGEs contributing to renal and vascular complications [42]. Nevertheless, it remains to be determined the relative importance of these exogenous sources of AGEs in the pathogenesis of diabetic complications. Advanced glycation end products accumulate within the various organs that are damaged in diabetes, what is accelerated by hyperglycemia. The intermolecular collagen cross-linking caused by AGEs leads to diminished arterial and myocardial compliance and increased vascular stiffness, phenomena that are considered to partly explain the increase in diastolic dysfunction and systolic hypertension seen in diabetic subjects [43]. Advanced glycation end products accumulate in most sites of diabetes complications, including the kidney, retina, and atherosclerotic plaques [44, 45]. Advanced glycation end products have been measured and reported to be linked to the sustained effects of prior glycemic control on the subsequent development of vascular complications. Oxidative stress may play an important role in the development of complications in diabetes such as lens cataracts, nephropathy, and neuropathy. glycation reactions, especially Maillard reactions, occurring in vivo as well as in vitro and are associated with the chronic complications of diabetes mellitus, aging, and age-related diseases by increases in oxidative chemical modification of lipids, DNA, and proteins [46]. In particular, long-lived proteins such as lens crystallines, collagens, and hemoglobin may react with reducing sugars to form AGEs. Recently, we found a novel type of AGE, named MRX, and we found that MRX is a good biomarker for detecting oxidative stress produced during Maillard reaction [47]. Lipid peroxidation reaction in hyperglycemia and hexanoyl modification formed by the reaction of oxidized lipids and proteins might be important for oxidative stress development. Indeed, the hexanoyl lysine (HEL) moiety in proteins, the earlier and stable markers for lipid peroxidation–derived protein [48], has been identified in oxidized LDL and erythrocytes of patients with type 1 diabetes [49]. On the other hand, macrophages and neutrophils play an important role in oxidative stress during hyperglycemia, and it has been determined that oxidatively modified tyrosines are a good biomarker for the occurrence of oxidative stress at an early stage. In this point, the interaction of AGEs with RAGE in endothelial and inflammatory cells induces intracellular generation of ROS, mainly mitochondrial electron transport chain, NADPH oxidase, xanthine oxidase, and arachidonic acid metabolism [50]. Omori et al [51] describe the kinetics of p47phox activation comparing neutrophils from diabetic and healthy subjects, suggesting that hyperglycemia increases AGE prime neutrophils. In turn, this increases the oxidative stress through the induction of the p47phox translocation to the cell membrane, and preassembly with p22phox by stimulating a RAGEERK1/2 pathway. Several reports have linked ROS with intracellular and extracellular inflammatory signals, which induce the signal transduction from RAGE to NF-κB [52, 53].

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Each of these pathways is closely linked to AGE binding to RAGE, because blockade of the receptor with either anti–RAGE IgG or excess soluble RAGE prevents their activation [54]. Therefore, the inhibition of AGE formation, blockade of the AGE-RAGE interaction, and suppression of RAGE expression or its downstream pathways may be a novel therapeutic strategy for the treatment of vascular complications in diabetes.

2.4. Role of Oxidative Stress in Diabetes 2.4.1. Clinical Evidence for Glucose Toxicity The term glucotoxicity (or glucose toxicity) refers to a phenomenon responsible for the pathologic changes on cellular function and structure in tissues throughout the body, due to the adverse effects of elevated blood glucose levels. The dimension of time is essential to the toxic effects of glucose; they are best understood in the context of chronic, time-related elevations of blood glucose over many months and years rather than days. Because blood glucose levels in nondiabetic people rise postprandially, it seems unlikely that short periods of elevated blood glucose are significantly toxic to cells. The concept of glucose toxicity at the level of the pancreatic islet β-cell is more relevant to type 2 diabetes than to type 1 diabetes because, as mentioned above, patients with type 2 diabetes typically retain functional β-cells for many years after the onset of the disease. Even though optimal medical management for type 2 diabetes regulates fasting glucose levels, most patients continue having abnormally elevated glucose levels postprandially. Such patients are continually in double risk because they have a disease that both decreases β-cell function and has an outcome (hyperglycemia) that continually damages the remaining β-cells. For many years it has been suggested that patients with diabetes undergo chronic oxidative stress. This can be appreciated by measurements of biomarkers for oxidative stress in patients with type 2 diabetes using various laboratory techniques, including highperformance liquid chromatography, gas chromatography/mass spectrometry, and immunostaining of pancreatic biopsies. Elevated oxidants and markers for oxidative tissue damage, such as hydroperoxides, oxidation of DNA bases, 8-epi-prostaglandin F2α, and 8hydroxy-2’-deoxyguanosine, have been reported in patients with diabetes [55, 56, 57]. Moreover, therapy with sulfonylureas, which low blood glucose levels in diabetic patients, has been associated with an increase in red blood cell glutathione [58], which enhances intracellular antioxidant defense mechanisms. Coincidentally, several conventional antihyperglycemic drugs can also show antioxidant activities [59, 60]. Activity of the ratelimiting enzyme for glutathione synthesis, γ-glutamylcysteine ligase, was reported to increase with improved glycemic control [61]. Using human isolated islets, it has been reported that exposure to high glucose concentrations increases intra islet levels of peroxide [62]. This increase was blocked by mannoheptulose, which prevents glucose metabolism by the β-cell. Recent studies suggested that subclinical cardiovascular disease, including complications in diabetes, is associated with oxidative damage and precedes future cardiovascular disease [63, 64]. Blood levels of glucose, D-dimer, glutathione and total cholesterol contribute

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significantly to a diabetic oxidative damage, constituting a panel of biomarkers that may be helpful in evidence-based pharmacological intervention with anti-aggregation and/or antioxidant agents against cardiovascular disease in diabetes. Actually, some clinical trials have suggested that non-insulin-dependent diabetes mellitus patients present increased lipid peroxidation, changes in antioxidative defense (decreased CuZnSOD activity in erythrocytes), and alterations in erythrocyte morphology compared with insulin-dependent diabetic patients [65, 66]. Diabetic patients with microvascular complications show, increased oxidative stress related-parameters such as malondialdehyde levels, together with decreased glutathione peroxidase and superoxide dismutase activities. These factors may contribute to the occurrence of micro vascular complications and mortality in non-insulindependent diabetes mellitus patients. 2.4.2. Experimental Studies in Animal Models of Diabetes Observations using clinical material obtained from humans that suggested a link between glucose toxicity and oxidative stress have stimulated a great deal of research in animal models of diabetes. The advantages of studying the streptozotocin-treated diabetic mouse and the manipulation of apoE as the preferred model to the streptozotocin-induced diabetic rat was first reported by Yamamoto et al. [67] . Nonetheless, there is a more extensive literature concerning the development of vascular disease in the streptozotocin-diabetic rat. For instance, in the aorta from this model it was reported a triphasic change in endothelial function enhanced at 1-week post-streptozotocin, unaltered at 1–2 weeks, and impaired at 8 weeks [68]. Kobayashi and Kamata [69], reported a decrease in aortic endothelial function 9 weeks after streptozotocin treatment and this was accompanied by an increase in oxidative stress, but no change in mRNA eNOS expression. In contrast, in rats treated for 2 weeks with streptozotocin, aortic eNOS mRNA and NADPH oxidase were increased and endothelial dysfunction was associated with a reduction in the bioavailability of NO as measured by electron spin resonance [70]. In the current study [71], it was also shown that biomarkers of oxidative stress were elevated in the aorta from the streptozotocin-treated groups at 16 weeks. Similar changes occur in the conduit vessels, what has also been reported for the small mesenteric vessels from the apoE−/− streptozotocin diabetic mouse [72]. Recent studies followed in ApoE-deficient (ApoE-/-) and glutathione peroxidase (GSH-Px) double-knockout (ApoE-/- GSH-Px-/-) mice diabetic with streptozotocin, showed that lack of functional GSHPx accelerates diabetes-associated atherosclerosis via up-regulation of proinflammatory and profibrotic pathways. These data establishe GSH-Px as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease [73]. A model of type 2 diabetes are Zucker diabetic fatty (ZDF) rats, showing an inadequate control to maintain normoglycemia. These animals are leptin receptor deficient and are characterized by postweaning development of marked obesity, hyperglycemia, and hypertriglyceridemia [74]. The chronic exposure of this animal type to high glucose levels over many months caused diminished insulin gene expression, insulin content, and glucoseinduced insulin secretion [75, 76]. These abnormalities were associated with decreased levels of two critical insulin promoter transcription factors [77, 78]. The ZDF rats became more hyperglycemic and they developed defects in these factors, as well as decreased insulin

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mRNA levels, insulin content, and glucose-induced insulin secretion in isolated islets [79]. When the animals were given troglitizone, a glucose- and triglyceride-lowering drug that is also an antioxidant, during the first 6–16 weeks of age, the development of hyperglycemia was substantially prevented. There exists in vivo evidence that the relentless progression of hyperglycemia in the animal had secondary glucotoxic effects on the remaining β-cells [80]. To ascertain whether these glucotoxic effects were due to oxidative stress, the animals were treated with two antioxidants, N-acetylcysteine (NAC) or aminoguanine (AG). These drugs were given to ZDF rats from the 6th to the 12th week of age. Both drugs prevented the rise in blood oxidative stress markers, including malondialdehyde and 4-hydroxy-2-nonenal. The drugs ameliorated the development of hyperglycemia, glucose intolerance, defective insulin secretion, decrements in β-cell insulin content, and insulin gene expression [81]. These studies strengthened the hypothesis that chronic oxidative stress is a major mechanism for the clinical phenomenon termed glucotoxicity of the pancreatic β-cell. Similar observations were made using another type 2 diabetics models, the db/db mouse, where antioxidant treatment NAC, vitamins C plus E, or both for 6 weeks revealed that the β-cell mass was significantly larger in the diabetic mice treated with the antioxidants than in the untreated mice. As a possible cause, the antioxidant treatment suppressed apoptosis in β-cell without changing its rate of proliferation, supporting the hypothesis that in chronic hyperglycemia, apoptosis induced by oxidative stress causes reduction of β-cell mass [82]. Recent studies confirm the importance of oxidative stress in diabetic vascular dysfunction [83, 84, 85]. These data reveal a different origin of ROS under basal conditions and during stimulation with contracting agents. The increased basal superoxide radical of the smooth muscle of diabetic rats appears to be derived from NADPH oxidase. The free radicals produced by NADPH oxidase cause the up-regulation of both constitutive and inducible COXs in the vascular smooth muscle cells [86]. These enzymes generate oxygen-derived free radicals and/or metabolites of arachidonic acid, which in turn cause depression of the contractile process but hyper-responsiveness of the cell membrane receptors of the vascular smooth muscle cells [87]. Therefore, the mechanisms determining an endothelial dysfunction are implicated in macro- and microvascular complications in diabetic patients, derived in part from an imbalance of vasoconstrictor prostanoids.

3. Prevention with Antioxidants 3.1. Clinical Trials In the last time, much attention has been focused on attenuation of oxidative stress by dietary antioxidants to assist in the prevention of diabetes mellitus. An increase in oxidizing response above a certain threshold produces, in the absence of a concomitant rise in antioxidant/reducing response, oxidative stress that is associated with complications in diabetes. In relation to this concept, the absence of complications in type 1 diabetes patients up to 5 years after onset of the disease may be associated with the oxidizing and reducing balance which needs to be maintained in order to prevent or delay the onset of oxidative stress [88]. The effective diabetic control involves evaluation of the oxidizing/antioxidant

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balance besides glycemic control. A simple technique involving reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye has been developed in order to determine quantitatively the antioxidant status of plasma [89]. Human plasma contains several antioxidant effectors including ascorbate, urate, α-tocopherol, albumin and protein sulphydryl groups [90]. Previously it has been found that oxidized human serum albumin, in contrast to normal albumin, decreases antioxidant activity and is able to trigger the oxidative burst of human neutrophils [91]. In this point, various studies show that plasma from healthy subjects exhibited a higher antioxidant status compared with plasma from diabetic patients, indicating that the latter may contain high levels of protein and oxidized lipids, possibly occasioned by oxidative stress present in diabetes [92, 93]. In several of its roles, selenium functions as a dietary antioxidant and thus has been studied for its possible role in chronic diseases [94]. Indeed, it could play an important role in diabetic complications, performing as a cofactor for antioxidant GSH-Px activity [95]. Kornhauser et al., [96] found that in type 2 diabetes mellitus patients with microalbuminuria showed a positive correlation with glucose, and a negative correlation with serum selenium and glutathione peroxidase, suggesting that this may be implicated in the diabetic nephropathy. In other recent study that supports the importance of impaired antioxidant status for the development of insulin resistance and type 2 diabetes [97], serum concentrations and dietary intakes of beta-carotene and alpha-tocopherol, independently, predicted insulin resistance and type 2 diabetes incidence during 27 years of follow-up in a community-based study of men. On the other hand, dietary supplementation with micronutrients may be a complement to classical therapies for preventing and treating diabetic complications. Supplementation is expected to be more effective when a deficiency in these micronutrients exists. Nevertheless, many clinical studies have reported beneficial effects in individuals without deficiencies, although several of these studies were short-term and had small sample sizes [98]. Recently, it was found that strong antioxidative activity in curcuminoids, the main yellow pigments in Curcuma longa (turmeric), have been used widely and for a long time in the treatment of sprain and inflammation in indigenous medicine. Curcumin (U1) is the main component of turmeric, and two minor components are also present as the curcuminoids. Curcuminoids were reported to possess antioxidant activity [99, 100] and inhibit the microsome-mediated mutagenicity of benzo(a)pyrene and 7,12-dimethyl-benz(a)anthracene, and it was also reported that U1 acts as a strong inhibitor of tumor promotion. This effect can be explained as roughly parallel to the relative antioxidant activity [101, 102]. In the case of the antioxidant supplement, various studies support the preventive effects on metabolic and clinical complications of diabetes, mainly in type 1 diabetes [103, 104, 105]. This clinical trial identifies positive effects of these nutrients on various outcome measures relating insulin resistance and cardiovascular factors [106]. However, the potential role of these agents, such as alpha-lipoic acid, chromium, folic acid, isoflavones, magnesium, pycnogenol, selenium, vitamin C, vitamin E, and zinc in the treatment of type 2 diabetes are not well characterized. This can be mainly due to the lack of rigor in applying the inclusion criteria and in excluding those concomitant pathologies that would be interfering with oxidative stress-related parameters. In the case of vitamin E, it is reported to reduce oxidative stress at levels of 200 mg day or more, especially in patients with renal disease [107] and coronary artery disease [108]. Accordingly, Engelen et al., [109] developed a clinical trial

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where the combination of micronized fenofibrate 200 mg/day and vitamin E 400 IU/day tended to increase the resistance of non-HDL lipoproteins to copper-mediated oxidation. Vitamin E alone administration, decreased the oxidation of non-HDL lipoproteins as shown by a reduction of thiobarbituric acid reactant substances formation. This protective effect of vitamin E tended to be enhanced by micronized fenofibrate. The supplement of vitamin C also showed an attenuation of oxidative damage in diabetes mellitus patients [110, 111]. It is known that vitamin C at pharmacologic doses decreases sorbitol accumulation, product that contributes to the progression of chronic diabetic complications. One the first trials reported of vitamin C supplements intake of 100 or 600 mg daily for 58 days in young adults with insulin-dependent diabetes mellitus and nondiabetic adults [112]. This supplement diminished significantly the sorbitol accumulation in the erythrocytes of diabetics, displaying low toxicity. This accounted for the efficacy of vitamin C over pharmaceutical aldose reductase inhibitors. Other study, a placebo-controlled trial, tested the hypothesis that oral prophylaxis with vitamin C attenuates rest and exerciseinduced free radical-mediated lipid peroxidation in type 1 diabetes mellitus [113]. Venous blood samples were obtained at rest, after a maximal exercise challenge and before and 2h after oral ingestion of 1g ascorbate or placebo. Diabetic patients exhibited an elevated concentration of lipid peroxidation associated with a depression of retinol and lycopene. Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in both groups and attenuated the exercise-induced oxidative stress response compared with healthy individuals. Finally, flavonoids are described to exert a large array of biological activities, which are mostly ascribed to their radical-scavenging, metal chelating and enzyme modulation ability. Most of these evidences have been obtained by in vitro studies on individual compounds and at doses largely exceeding dietary doses [114]. Various authors have found that flavonoid consumption has a positive effect on insulin resistance and cardiovascular outcome measures, but only when combined with soy proteins [115, 116]. Indeed, antioxidant effects have been measured on serum and macrophages, which could contribute to attenuation of atherosclerosis development in non-insulin dependent diabetes mellitus patients [117].

3.2. Experimental Studies of Antioxidants in Diabetes There exists evidence that the over expression of antioxidants such as Cu/Zn superoxide dismutase [118], catalase [119], thioredoxin [120], and metallothionein [121] targeted at βcells counteracts the development of type 1 diabetes. Since the pancreas has been shown to have low levels of the antioxidant enzymes superoxide dismutase and catalase [122], these findings suggest that oxidative stress plays a key role in the pathogenesis of diabetes [123]. Indeed, nitric oxide (NO) and ROS induced by inflammatory cytokines such as interleukin1β, TNF-α, and interferon-γ are considered to be crucial mediators of β-cell death [124, 125]. This is supported by several studies showing that pretreatment with antioxidants before the injection of streptozotocin attenuates the development of hyperglycemia and insulitis in the multiple low-dose streptozotocin models [126, 127, 128]. In the case of vitamin E, chronic effect of supplementation in addition to insulin can have additive protective effects against

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deterioration of renal function in rat model of diabetic nephropathy [129]. In addition, kidney tissue levels of malondialdehyde and inducible NO synthase (iNOS), and reduced serum glutathione peroxidase improved towards control levels with vitamin E administration. These effects could be associated with protection of vessel structure and function in the damage of microvascular complications [130]. Similar experimental models in STZ-treated rats have been used to assess the effect of short-term antioxidant therapy based on oral vitamins C and E administration, with similar antioxidant response [131, 132]. In the case of vitamin C, the use for attenuating oxidative injury might become a useful adjunct to prevent albuminuria and renal sclerosis in rat diabetic nephropathy [133]. It was demonstrated that ascorbic acid directly modulates contractile responses of diabetic rat aortas, likely through mechanisms in part dependent of preservation of endothelium-derived NO [134]. In this point, vitamin C may act as a direct ROS scavenger in endothelium; resulting in increased availability of the eNOS cofactor tetrahydrobiopterin, thereby enhancing eNOS activity [135]. With respect to ROS sources, Yamamoto et al., [67] described for the fisrt time increased oxidative stress linked to altered expression of NADPH oxidase and eNOS at an early stage of the induction of diabetes with streptozotocin in the apoE−/− mouse. In addition, expression of both mitochondrial SOD initially (at 4-week post-streptozotocin) and then the cytosolic SOD (at 8-week post-streptozotocin) increased in the mesenteric arteries perhaps reflecting a compensatory response to the increase in oxidative stress. However, SOD1 and SOD2 levels were not different when the streptozotocin-treated group were compared to the control group at 16 weeks again, suggesting that the reported changes in SOD are linked to the initiation of type 1 diabetes following treatment with streptozotocin and the onset of diabetes-related vascular dysfunction. Nevertheless, expression of eNOS mRNA and protein remained significantly elevated at 16 weeks. An enhanced expression of eNOS may contribute to vascular dysfunction and accelerated atherogenesis as reported in the apoE−/− mouse [136]. On the other hand, Ding et al. [137] have reported that in the small mesenteric arteries from the streptozotocin-diabetic apoE−/− mouse the component of acetylcholine-mediated relaxation attributed to NO from streptozotocin-treated diabetic apoE−/− mice was enhanced in comparison to the non-diabetic control apoE−/− mouse. This suggested that the ability of eNOS to generate NO is not compromised in this type 1 diabetic model, possibly reflecting an enhanced synthesis of tetrahydrobiopterin that has also been reported in the vasculature of the apoE−/− mouse [138]. The eNOS expression and endothelial function in the streptozotocin-type 1 apoE−/− mouse differs from that reported for the db/db mouse model of type 2 diabetes. In small mesenteric arteries from these mice there was, a complete absence of the contribution from NO to endothelium-dependent relaxation, elevated oxidative stress, and no change in eNOS mRNA or protein expression [139]. In the case of the NADPH oxidase, an increase in gp91phox expression has also been linked to neovascularization following tissue ischemia [140]. In the aorta from the streptozotocin-rat model of type 1 diabetes [141], reported that 8 weeks after treatment with streptozotocin Nox1 protein expression was doubled, but the expression of nox4 was unchanged and acetylcholine-mediated vasodilatation and NO generation was decreased despite an increase in eNOS protein. In this view, there are conflicting reports on the effects of NOS inhibitors on type 1 diabetes in animal models [142, 143]. Papaccio et al., [144] reported that multiple low-dose

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streptozotocin treatment did not stimulate NO production at the islet level, although superoxide dismutase activity was decreased. These data suggest that NO itself may have no major role in multiple low-dose streptozotocin-induced diabetes. However, Lenzen [145] and Mabley et al. [146] have demonstrated that peroxynitrite plays a role in the pathogenesis of islet cell dysfunction and the destruction associated with the multiple low-dose streptozotocin model of type 1 diabetes. Recently, it has been demonstrated that edaravone, a potent scavenger of hydroxyl and peroxyl radicals, protects against NO-induced cytotoxicity in cultured astrocytes, although edaravone does not affect the release of NO or its metabolism [147]. In addition, edaravone protects against S-nitroso-N-acetyl-DL-penicillamine, a NO donor that induces cytotoxicity in the rat pancreatic β-cell line INS-1. These findings suggest that NO plays a role in the pathogenesis of diabetes and that edaravone may attenuate multiple low-dose streptozotocininduced diabetes by inhibiting a NO-mediated mechanism. Considering the NADPH oxidase as a potential pharmacological target in experimental diabetic model, some evidences support that the experimental inhibition of this enzyme is associated to a potential benefit in diminishing the complications associated with the microvascular [148] and macrovascular damage [149]. In this point, some drugs with antioxidant properties have been used as coadjutant therapy in vascular disorders associated with diabetes progression such as β-blockers [150], ACE inhibitors [151], and statins. These drugs normalizes endothelial function and reduce oxidative stress in diabetes by inhibiting the activation of the vascular NADPH oxidase, thereby preventing eNOS uncoupling due to an up-regulation of the key enzyme of tetrahydrobiopterin synthesis [152]. Other of their properties involves an improvement of the endothelium-dependent vasodilatation [153], lipid-independent antioxidant [154] and anti-inflammatory effects [155]. The proposed mechanism for these pleiotropic effects, even at low-dose, involves the inhibition of inflammatory intracellular pathway via ERK1/2/NF-kappaB-pathway [156], and inactivation sources of ROS such as NADPH oxidase [157]. Both of these effects have been demonstrated sufficiently to improve endothelial function under experimental diabetic conditions.

3.3. Role of Antioxidants in Reducing the Advanced Glycation End Product Formation Aminoguanidine was one of the first inhibitors of AGE formation studied [158, 159], and is thought to act as a nucleophilic trap for carbonyl intermediates. In animal studies, aminoguanidine has prevented a wide range of diabetic vascular complications [160, 161, 162]. In clinical trials, it has been found a reduction in AGE-hemoglobin independent of HbA1c lowering [163, 164]. Placebo-controlled clinical trials have been assayed with aminoguanidine in types 1 and 2 diabetes examining renal outcomes [165, 166]. Reductions in proteinuria and decreased progression of retinopathy were observed, although the study did not demonstrate a statistically significant beneficial effect on the progression of nephropathy. Further clinical evaluation of this agent has been limited due to long-term toxicity. Indeed, some patients

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have developed antimyeloperoxidase and antineutrophil antibodies [167], and also glomerulonephritis [166]. Pyridoxamine is a derivative of vitamin B6. It prevents the degradation of proteinamadori intermediates to protein-AGE products. In murine models it has reduced hyperlipidemia and prevented AGE formation [168, 169]. Thomas et al. [170] showed that pyridoxamine antagonizes angiotensin II-induced elevation in serum and renal AGEs, prevents renal hypertrophy, and decreases salt retention in experimental models. Pyridoxamine also prevents diabetes-induced retinal vascular lesions. Some preliminary clinical trials with this agent have been performed [171]. Benfotiamine, a lipid-soluble thiamine (vitamin B1) derivative, prevents activation of three major pathways of hyperglycaemic damage (hexosamine pathway, intracellular AGE formation, and the diacylglycerol-protein kinase C pathway) by increasing the activity of transketolase, the rate-limiting enzyme of the nonoxidative branch of the pentose phosphate pathway [172]. In animal studies, high-dose thiamine and benfotiamine therapy increased transketolase expression in renal glomeruli, and inhibited the development of microalbuminuria and diabetes-induced hyperfiltration [173]. Benfotiamine has improved nerve conduction velocity in the peroneal nerve [174] in diabetic patients. Accordingly, a short 3-week clinical study showed alleviation of painful neuropathy [175]. Nevertheless, long-term human data are still lacking. Recent studies also show that benfotiamine prevents macrovascular and microvascular endothelial dysfunction and oxidative stress after a highAGE meal [176]. Methylene bis 4,4-(2 chlorophenylureido phenoxyisobutyric acid) (LR-90) has been investigated in a number of animal studies by Figarola et al., [177] The compound LR-90 inhibited albuminuria, reduced serum creatinine concentration, and circulating AGE levels in diabetic rats without any effect on glycemic control. Also, LR-90 prevented glomerulosclerosis and collagen deposition in association with reduced glomerular AGE accumulation. The effect of LR-90 is also currently being tested on macrovascular complications in a range of animal studies. Interestingly, LR-90 has recently inhibited the expression of proinflammatory molecules stimulated by RAGE pathway in human monocytes[178], suggesting that this agent has novel antiinflammatory properties with protective effects against diabetic vascular complications. There are plans for this agent to be further evaluated in the clinical context. Another therapeutic approach that is being considered involves soluble RAGE (sRAGE) that blocks AGEs from binding to RAGE. It remains to be fully elucidated if sRAGE acts whether as an antagonist inhibiting RAGE dependent signaling pathways or through binding various RAGE ligands such as AGEs, thus preventing these putative proinflammatory molecules from acting on other receptors (e.g scavenger receptors) to promote end-organ injury [179]. Studies with RAGE knockout mice that do not express sRAGE or full-length RAGE suggest that the key mechanism of sRAGE action is via inhibition of RAGE dependent phenomena. In the next few years, either sRAGE or possibly a nonpeptide RAGE antagonist will be examined clinically, although it is possible that nondiabetic diseases may be the focus of the RAGE clinical development program. The pathophysiological crosstalk between the AGE-RAGE system and angiotensin II has also been associated with diabetic microangiopathy in various diabetics’ models [180].

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Angiotensin convertase enzyme inhibitors as well as angiotensin II antagonists appear to decrease the formation of AGE, as assessed in a series of in vitro studies as well as in animal models of diabetes [181, 182]. Furthermore, ACE inhibitors, based on in vitro, preclinical, and clinical studies, appear to promote surface receptor AGE expression, thus providing an additional mechanism to inhibit AGE induced organ injury [183]. The explanation of these effects may be supported by a down-regulation of RAGE mRNA levels in a dose-dependent manner, demonstrated in some in vitro models. Indeed, ACE inhibitors may act as an antiinflammatory agent against AGE by suppressing RAGE expression via PPAR-gamma activation in the liver and may play a protective role in vascular injury in diabetes [184] (table 8-1). Table 8-1. The antioxidant interventions targeting the advanced glycation end products pathway Compound

Mechanism

ACE inhibitors

↓ Angiotensin II NADPH inactivation

Aminoguanidine

Inhibition AGE formation

Pyridoxamine LR-90 Anti-RAGE sRAGE

Inhibition AGE formation ↓ Cholesterol ↓ Oxidative stress ECM fibrosis RAGE blockade

Effects in humans ↓nephropathy, retinopathy Yoshida et al., 2006 Coughlan et al., 2007 ↓nephropathy, retinopathy Bolton et al., 2004 ↓retinopathy Degenhardt et al., 2002 ↓nephropathy Figarola et al., 2007 ↓Nephropathy Kunt et al., 2004 Thallas-Bonkeet al., 2004

ACE: Angiotensin convertase-enzyme; NADPH: nicotinamide adenine dinucleotide phosphate-oxidase; RAGE: Receptor advanced glycosilation end products; LR-90: Methylene bis 4,4--(-2 chlorophenylureido phenoxyisobutyric acid).

4. Conclusions and Perspectives Diabetes mellitus is a chronic disease caused by an impairment of β-cells function and survival, giving rise to the development of hyperglycemia. Consequently, the exposure to high blood glucose levels over many years leads to adverse structural and functional changes in tissues, a phenomenon termed glucose toxicity. The finding that diabetic patients have increased clinical biomarkers for oxidative stress and tissue damage could suggest a possible linkage between glucose toxicity and oxidative stress, what has been supported by experimental studies. Under diabetic conditions, ROS are produced through several processes such as the nonenzymatic glycosylation reaction, alterations in the electron transport chain in mitochondria, and the hexosamine pathway. Oxidative stress and consequent activation of the inflammatory and apoptotic pathways are likely involved in progression of pancreatic β-cell dysfunction found in diabetes type 1. Antioxidant treatment can protect β-cells against

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glucose toxicity and thus exert beneficial effects reported from clinical and biochemical assays. In the case of type 2 diabetes, ROS are accepted as major factors in the onset and development of their macrovascular complications. The underlying mechanism of this deleterious effect involves an inactivation of the signaling pathway between the insulin receptor and the glucose transporter system leading to the onset of insulin resistance in this setting. When comparing metabolic pathways of ROS production in the β-cell, it has been suggested that secretagogues causing increased insulin secretion can also lead to increased ROS production, via mitochondrial and NADPH oxidase mechanisms. The development of oxidative stress could be due not solely to the relatively of NADPH oxidase activity, but also to the low activity of antioxidant enzymes found in the islet β-cell. Therefore, together with scavenging ROS with antioxidant substances, it is plausible the design of future therapies based on targeting NADPH oxidase in the islet, thereby causing a diminution of ROS production. These interventions may be beneficial for maintaining β-cell integrity in the difficult environment of nutrient oversupply and immune challenge. The biochemical process of advanced glycation appears to be enhanced in the diabetic patients as a result of not only hyperglycemia but also due to other stimuli such as oxidative stress and increased free fatty acids. These might generate a heterogeneous group of chemical moieties that appear to induce directly and indirectly the development and progression of vascular complications. A range of pharmacological strategies, predominately being examined in preclinical contexts, appears to show great promise in reducing AGE induced injury by interfering with either the accumulation of AGE ligands or interrupting the AGERAGE interaction. It is anticipated that over the next few years, findings from clinical studies will assist clinicians in determining the relevance of targeting advanced glycation as an approach to reducing diabetic complications. Finally, a sufficient supply of dietary antioxidants may prevent or delay diabetes complications, including renal and neural dysfunction by providing protection against oxidative stress, although the detailed examination of protective mechanisms is uncertain. Therefore, it is extremely important that future clinical trials exert strict clinical criteria of selection, sources and doses of antioxidants.

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[119] Xu B, Moritz JT, Epstein PN. Overexpression of catalase provides partial protection to transgenic mouse beta cells. Free Radic. Biol. Med. 1999;27:830-837. [120] Hotta M, Tashiro F, Ikegami H, Niwa H, Ogihara T, Yodoi J, Miyazaki J.Pancreatic beta cell-specific expression of thioredoxin, an antioxidative and antiapoptotic protein, prevents autoimmune and streptozotocin-induced diabetes. J. Exp. Med. 1998;188:1445-1451. [121] Chen H, Carlson EC, Pellet L, Moritz JT, Epstein PN. Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes. Diabetes. 2001;50:2040-2046. [122] Lenzen S, Drinkgern J, Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic. Biol. Med. 1996;20:463-466. [123] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell. Biol. 2007;39:44-84. [124] Lakey JR, Suarez-Pinzon WL, Strynadka K, Korbutt GS, Rajotte RV, Mabley JG, Szabó C, Rabinovitch A. Peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells. Lab. Invest. 2001;81:1683-1692. [125] Rabinovitch A, Suarez-Pinzon WL. Cytokines and their roles in pancreatic islet betacell destruction and insulin-dependent diabetes mellitus. Biochem. Pharmacol. 1998;55:1139-1149. [126] Mabley JG, Rabinovitch A, Suarez-Pinzon W, Haskó G, Pacher P, Power R, Southan G, Salzman A, Szabó C. Inosine protects against the development of diabetes in multiple-low-dose streptozotocin and nonobese diabetic mouse models of type 1 diabetes. Mol. Med. 2003;9:96-104. [127] Song Y, Song Z, Zhang L, McClain CJ, Kang YJ, Cai L. Diabetes enhances ipopolysaccharide-induced cardiac toxicity in the mouse model. Cardiovasc. Toxicol. 2003;3:363-372. [128] Szabó C, Mabley JG, Moeller SM, Shimanovich R, Pacher P, Virag L, Soriano FG, Van Duzer JH, Williams W, Salzman AL, Groves JT. Part I: pathogenetic role of peroxynitrite in the development of diabetes and diabetic vascular complications: studies with FP15, a novel potent peroxynitrite decomposition catalyst. Mol. Med. 2002;8:571-580. [129] Haidara MA, Mikhailidis DP, Rateb MA, Ahmed ZA, Yassin HZ, Ibrahim IM, Rashed LA. Evaluation of the effect of oxidative stress and vitamin E supplementation on renal function in rats with streptozotocin-induced Type 1 diabetes. J. Diabetes. Complications. 2008. [Epub ahead of print] [130] Emekli U, Tuncer S, Kabakas F, Aydin A, Arinci A, Bilgic B, Haklar G. The effect of short- versus long-term administration of alpha tocopherol on the survival of random flaps in experimental diabetes mellitus. J. Diabetes. Complications. 2004;18:249-257. [131] Cameron NE, Cotter MA. Effects of antioxidants on nerve and vascular dysfunction in experimental diabetes. Diabetes Res. Clin. Pract. 1999;45:137-146.

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[132] Rupérez FJ, García-Martínez D, Baena B, Maeso N, Cifuentes A, Barbas C, Herrera E. Evolution of oxidative stress parameters and response to oral vitamins E and C in streptozotocin-induced diabetic rats. J. Pharm. Pharmacol. 2008;60:871-878. [133] Lee EY, Lee MY, Hong SW, Chung CH, Hong SY. Blockade of oxidative stress by vitamin C ameliorates albuminuria and renal sclerosis in experimental diabetic rats. Yonsei Med. J. 2007;48:847-855. [134] Ajay M, Mustafa MR. Effects of ascorbic acid on impaired vascular reactivity in aortas isolated from age-matched hypertensive and diabetic rats. Vascul. Pharmacol. 2006;45:127-133. [135] Baker TA, Milstien S, Katusic ZS. Effect of vitamin C on the availability of tetrahydrobiopterin in human endothelial cells. J. Cardiovasc. Pharmacol. 2001;37:333-338. [136] Kawashima S, Yokoyama M. Dysfunction of endothelial NO synthase and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2004;24:998-1005. [137] Ding H, Hashem M, Wiehler WB, Lau W, Martin J, Reid J, Triggle C. Endothelial dysfunction in the streptozotocin-induced diabetic apoE-deficient mouse. Br. J. Pharmacol. 2005;146:1110-1118. [138] d'Uscio LV, Katusic ZS. Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice. Am. J. Physiol. Heart Circ. Physiol. 2006;290:H2466H2471. [139] Pannirselvam M, Simon V, Verma S, Anderson T, Triggle CR. Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br. J. Pharmacol. 2003;140:701-706. [140] Tojo T, Ushio-Fukai M, Yamaoka-Tojo M, Ikeda S, Patrushev N, Alexander RW. Role of gp91phox (Nox2)-containing NAD(P)H oxidase in angiogenesis in response to hindlimb ischemia. Circulation. 2005;111:2347-2355. [141] Wendt MC, Daiber A, Kleschyov AL, Mülsch A, Sydow K, Schulz E, Chen K, Keaney JF Jr, Lassègue B, Walter U, Griendling KK, Münzel T. Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4. Free Radic. Biol. Med. 2005;39:381-391. [142] Papaccio G, Esposito V, Latronico MV, Pisanti FA. Administration of a NO synthase inhibitor does not suppress low-dose streptozotocin-induced diabetes in mice. Int. J. Pancreatol. 1995;17:63-68. [143] McCabe C, O'Brien T. The rational design of beta cell cytoprotective gene transfer strategies: targeting deleterious iNOS expression. Mol. Biotechnol. 2007;37:38-47. [144] Papaccio G, Pisanti FA, Latronico MV, Ammendola E, Galdieri M. Multiple low-dose and single high-dose treatments with streptozotocin do not generate nitric oxide. J. Cell. Biochem. 2000;77:82-91. [145] Lenzen S. Oxidative stress: the vulnerable beta-cell. Biochem. Soc. Trans. 2008;36:343-347. [146] Mabley JG, Southan GJ, Salzman AL, Szabó C. The combined inducible nitric oxide synthase inhibitor and free radical scavenger guanidinoethyldisulfide prevents multiple

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low-dose streptozotocin-induced diabetes in vivo and interleukin-1beta-induced suppression of islet insulin secretion in vitro. Pancreas. 2004 Mar;28(2):E39-E44. [147] Kawasaki T, Kitao T, Nakagawa K, Fujisaki H, Takegawa Y, Koda K, Ago Y, Baba A, Matsuda T. NO-induced apoptosis in cultured rat astrocytes: protection by edaravone, a radical scavenger. Glia. 2007;55:1325-1333. [148] Danesh FR, Kanwar YS. Modulatory effects of HMG-CoA reductase inhibitors in diabetic microangiopathy FASEB J. 2004;18:805-815. [149] Hamilton CA, Brosnan MJ, Al-Benna S, Berg G, Dominiczak AF. NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels. Hypertension. 2002;40:755-762. [150] Bank AJ, Kelly AS, Thelen AM, Kaiser DR, Gonzalez-Campoy JM. Effects of carvedilol versus metoprolol on endothelial function and oxidative stress in patients with type 2 diabetes mellitus. Am. J. Hypertens. 2007;20:777-783. [151] Yazici D, Yavuz DG, Unsalan S, Toprak A, Yüksel M, Deyneli O, Aydin H, Tezcan H, Rollas S, Akalin S. Temporal effects of low-dose ACE inhibition on endothelial function in Type 1 diabetic patients. J. Endocrinol. Invest. 2007;30:726-733. [152] Wenzel P, Daiber A, Oelze M, Brandt M, Closs E, Xu J, Thum T, Bauersachs J, Ertl G, Zou MH, Förstermann U, Münzel T.Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus. Atherosclerosis. 2008;198:65-76. [153] Koh KK, Quon MJ, Han SH, Lee Y, Ahn JY, Kim SJ, Koh Y, Shin EK. Simvastatin improves flow-mediated dilation but reduces adiponectin levels and insulin sensitivity in hypercholesterolemic patients. Diabetes Care. 2008;31:776-782. [154] Rubba P. Effects of atorvastatin on the different phases of atherogenesis. Drugs. 2007;67:17-27. [155] Chen YH, Lin SJ, Chen YL, Liu PL, Chen JW. Anti-inflammatory effects of different drugs/agents with antioxidant property on endothelial expression of adhesion molecules. Cardiovasc. Hematol. Disord. Drug Targets. 2006;6:279-304. [156] Riad A, Du J, Stiehl S, Westermann D, Mohr Z, Sobirey M, Doehner W, Adams V, Pauschinger M, Schultheiss HP, Tschöpe C. Low-dose treatment with atorvastatin leads to anti-oxidative and anti-inflammatory effects in diabetes mellitus. Eur. J. Pharmacol. 2007;569:204-211. [157] Chen W, Pendyala S, Natarajan V, Garcia JG, Jacobson JR Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition. Am. J. Physiol. Lung Cell Mol. Physiol. 2008;295:L575-L583. [158] Hammes HP, Ali SS, Uhlmann M, Weiss A, Federlin K, Geisen K, Brownlee M. Aminoguanidine does not inhibit the initial phase of experimental diabetic retinopathy in rats. Diabetologia. 1995;38:269–273. [159] Kang KS, Yamabe N, Kim HY, Yokozawa T. Role of maltol in advanced glycation end products and free radicals: in-vitro and in-vivo studies. J. Pharm. Pharmacol. 2008;60:445-452. [160] Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, Jerums G. Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes. 1991;40:1328–1334

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[161] Edelstein D, Brownlee M. Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine. Diabetes. 1992;41:26–29. [162] Kazachkov M, Chen K, Babiy S, Yu PH. Evidence for in vivo scavenging by aminoguanidine of formaldehyde produced via semicarbazide-sensitive amine oxidasemediated deamination. J. Pharmacol. Exp. Ther. 2007;322:1201-1207. [163] Bucala R, Vlassara H. Advanced glycosylation end products in diabetic renal and vascular disease. Am. J. Kidney Dis. 1995;26:875–888. [164] Chang KC, Tseng CD, Wu MS, Liang JT, Tsai MS, Cho YL, Tseng YZ. Aminoguanidine prevents arterial stiffening in a new rat model of type 2 diabetes. Eur. J. Clin. Invest. 2006;36:528-535. [165] Freedman BI, Wuerth JP, Cartwright K, Bain RP, Dippe S, Hershon K, Mooradian AD, Spinowitz BS. Design and baseline characteristics for the aminoguanidine Clinical Trial in Overt Type 2 Diabetic Nephropathy (ACTION II). Control Clin. Trials. 1999;20:493–510. [166] Bolton WK, Cattran DC, Williams ME, Adler SG, Appel GB, Cartwright K, Foiles PG, Freedman BI, Raskin P, Ratner RE, Spinowitz BS, Whittier FC, Wuerth JP, ACTION I Investigator Group 2004 Randomized trial of an inhibitor of formation of advanced glycation end products in diabetic nephropathy. Am. J. Nephrol. 2004;24:32–40. [167] Whittier F, Spinowitz B, Wuerth JP. Pimagedine safety profile in patients with type 1 diabetes. J. Am. Soc. Nephrol. 1999;10:184A (Abstract A0941). [168] Degenhardt TP, Alderson NL, Arrington DD, Beattie RJ, Basgen JM, Steffes MW, Thorpe SR, Baynes JW. Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat. Kidney Int. 2002:61:939–950. [169] Stitt A, Gardiner TA, Alderson NL, Canning P, Frizzell N, Duffy N, Boyle C, Januszewski AS, Chachich M, Baynes JW, Thorpe SR. The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes. Diabetes. 2002;51:2826–2832. [170] Thomas MC, Tikellis C, Burns WM, Bialkowski K, Cao Z, Coughlan MT, JandeleitDahm K, Cooper ME, Forbes JM. Interactions between rennin angiotensin system and advanced glycation in the kidney. J. Am. Soc. Nephrol. 2005;16:2976–2984.. [171] Williams ME, Bolton WK, Khalifah RG, Degenhardt TP, Schotzinger RJ, McGill JB. Effects of pyridoxamine in combined phase 2 studies of patients with type 1 and type 2 diabetes and overt nephropathy. Am. J. Nephrol. 2007;27:605–614. [172] Hammes HP, Du X, Edelstein D, Taguchi T, Matsumura T, Ju Q, Lin J, Bierhaus A, Nawroth P, Hannak D, Neumaier M, Bergfeld R, Giardino I, Brownlee M. Benfotiamine blocks three major pathways of hyperglycaemic damage and prevents experimental diabetic retinopathy. Nat. Med. 2003;9:294–299. [173] Babaei-Jadidi R, Karachalias N, Ahmed N, Battah S, Thornalley PJ. Prevention of incipient diabetic nephropathy by high-dose thiamine and benfotiamine. Diabetes. 2003;52:2110–2120. [174] Stracke H, Lindemann A, Federlin K. A benfotiamine-vitamin B combination in treatment of diabetic polyneuropathy. Exp Clin Endocrinol Diabetes 1996;104:311-316

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[175] Haupt E, Ledermann H, Köpcke W. Benfotiamine in the treatment of diabetic polyneuropathy--a three-week randomized, controlled pilot study (BEDIP study). Int. J. Clin. Pharmacol. Ther. 2005;43:71-77. [176] Stirban A, Negrean M, Stratmann B, Gawlowski T, Horstmann T, Götting C, Kleesiek K, Mueller-Roesel M, Koschinsky T, Uribarri J, Vlassara H, Tschoepe D. Benfotiamine prevents macro- and microvascular endothelial dysfunction and oxidative stress following a meal rich in advanced glycation end products in individuals with type 2 diabetes. Diabetes Care. 2006;29:2064-2071. [177] Figarola JL, Scott S, Loera S, Tessler C, Chu P, Weiss L, Hardy J, Rahbar S. LR-90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats. Diabetologia. 2003 ;46:1140–1152. [178] Figarola JL, Shanmugam N, Natarajan R, Rahbar S. Anti-inflammatory effects of the advanced glycation end product inhibitor LR-90 in human monocytes. Diabetes. 2007;56:647–655. [179] Forbes JM, Thorpe SR, Thallas-Bonke V, Pete J, Thomas MC, Deemer ER, Bassal S, El-Osta A, Long DM, Panagiotopoulos S, Jerums G, Osicka TM, CooperME. Modulation of soluble receptor for advanced glycation end products by angiotensinconverting enzyme-1 inhibition in diabetic nephropathy. J. Am. Soc. Nephrol. 2005;16:2363–2372. [180] Bohlender J, Franke S, Sommer M, Stein G. Advanced glycation end products: a possible link to angiotensin in an animal model. Ann. N. Y. Acad. Sci. 2005;1043:681 4. [181] Forbes JM, Thomas MC, Thorpe SR, Alderson NL, Cooper ME. The effects of valsartan on the accumulation of circulating and renal advanced glycation end products in experimental diabetes. Kidney Int. Suppl. 2004;92:S105– S107. [182] Nangaku M, Miyata T, Sada T, Mizuno M, Inagi R, Ueda Y, Ishikawa N, Yuzawa H, Koike H, van Ypersele de Strihou C, Kurokawa K. Antihypertensive agents inhibit in vivo the formation of advanced glycation end products and improve renal damage in a type 2 diabetic nephropathy rat model. J. Am. Soc. Nephrol. 2003;14:1212–1222. [183] Coughlan MT, Thallas-Bonke V, Pete J, Long DM, Gasser A, Tong DC, Arnstein M, Thorpe SR, Cooper ME, Forbes JM. Combination therapy with the advanced glycation end product cross-link breaker, alagebrium, and angiotensin converting enzyme inhibitors in diabetes: synergy or redundancy? Endocrinology. 2007;148:886-895. [184] Yoshida T, Yamagishi S, Nakamura K, Matsui T, Imaizumi T, Takeuchi M, Koga H, Ueno T, Sata M. Telmisartan inhibits AGE-induced C-reactive protein production through downregulation of the receptor for AGE via peroxisome proliferator-activated receptor-gamma activation. Diabetologia. 2006;49:3094-3099.

In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter IX

Nonalcoholic Steatohepatitis Juan Gormaz1 and Ramón Rodrigo2 1

University of Chile Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile. Supported by FONDECYT, grant 1070948 2

Abstract Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver diseases characterized mainly by macrovesicular steatosis that occurs in the absence of alcoholic consumption. NAFLD is closely associated with comorbid conditions, such as obesity, dyslipidemia, and insulin resistance. It is a medical condition in which the liver is invaded with fat and excessive amounts of lipids are present within hepatocytes. There is increasing evidence to consider that fatty liver is the hepatic manifestation of the metabolic syndrome, a growing problem in the modern western world. NAFLD might worsen into a more serious condition, known as nonalcoholic steatohepatitis (NASH), in which fat accumulation is accompanied by an inflammatory process in the liver. The clinical relevance of these conditions is given by the high prevalence of NAFLD in the general population and to the possible evolution of NASH towards end-stage liver disease, including hepatocellular carcinoma, as well as the need for liver transplantation. The molecular mechanism whereby NASH might eventually lead to fibrosis, and severe cirrhosis in some patients, is a process associated with increased production and release of inflammatory mediators, such as nitric oxide (NO), cytokines, and reactive oxygen species (ROS) by the cells. Oxidative stress caused by increased ROS plays an important role in the pathogenesis of NASH. These reactive species would derive from mitochondria, cytochrome P-450 2E1, peroxisome, and iron overload in the liver with steatosis. Excessive ROS is considered to cause simple steatosis to progress to NASH. Regardless the origin of hepatic fat, it could produce a rise of hepatic free fatty acids. The latter, particularly the polyunsaturated ones, are closely linked to ROS generation by different pathways, including increased oxidation in different cellular organelles, disruption of mitochondria and endoplasmic reticulum, microsomal cytochrome P450

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Juan Gormaz and Ramón Rodrigo activation and ceramide formation. In addition, increased ROS production could derivate not only in hepatocyte cell death but also in the activation of liver resident cells, such as Kupffer, stellate and endothelial cells. This might enhance the original oxidative stress, inflammatory response and subsequent immune infiltration thus aggravating NASH. Up to date no absolute effective medical treatment is available for NASH patients. Therapy is predominantly aimed at controlling the comorbid conditions, such as obesity, insulin resistance, and dyslipidemia. However the major role of oxidative stress in the pathogenesis of NASH suggests that the antioxidant treatment would be an effective therapy. Hence, both several substances with different antioxidants mechanism and effects related with the redox balance have been assayed in small clinical trials. These agents have shown the ability of improve the outcome of patients, thus opening the door to new strategies to manage or treat this disease. This chapter provides the clinical and experimental evidence to support the role of oxidative stress in the pathophysiology of NAFLD and NASH, as well as the molecular bases promoting the development of mechanism-based therapeutic interventions, mainly clinical trials aimed to target specific pathways involved in the pathogenesis of NASH.

1. Introduction The liver plays an important role in lipid metabolism, including cholesterol, fatty acid and triglycerides synthesis, together with lipoproteins management [1]. Numerous factors, such as alcohol abuse, obesity, metabolic syndrome, hepatotoxic drugs, between others, can lead to metabolic disorders damaging liver structure and function. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of histologic findings ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis [2]. Nonalcoholic steatohepatitis is characterized histologically by the presence of hepatocyte ballooning, Mallory’s hyaline, inflammation, and fibrosis, which occur in the absence of excessive alcohol consumption [3].The diagnosis of hepatic steatosis and steatohepatitis or nonalcoholic steatohepatitis (NASH) is not yet possible without liver biopsy [4]. Nonalcoholic steatohepatitis is one of the most prevalent forms of chronic liver disease and it is estimated to affect nearly 4% of the United States population [5], approximately 1% of worldwide population [6]. Moreover, up to 20% of affected patients will develop cirrhosis [7]. In addition, near 70% of the cases of cryptogenic cirrhosis present characteristics of NASH [8]. This condition may represent the final step of NASH, which constitutes a lost of the typical necroinflammatory and steatotic characteristics, in up to 80% of patients [9, 10]. The 5- and 10-year survival in NASH has been calculated at 67% and 59%, respectively, although death often may be from comorbid pathologies. Currently it is accepted that NASH may lead to serious liver failure or hepatocellular carcinoma, [11, 12]. Within the North American population, the prevalence of NAFLD is nearly 20% [13, 14]; Japan and Italy have similar statistics [15, 16], becoming the most common cause of liver disease in Western countries [17]. Recent researches have demonstrated that NAFLD is closely related with obesity, dyslipidemia, insulin resistance, and has been described as the liver component of the metabolic syndrome [18]. For example, in obese population with a body mass index greater than 30 Kg/m2, the prevalence of NAFLD increases extremely, oscillating between 74% and

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90% [19, 20]. Epidemiological data also suggests a strong relation between insulin resistance and NAFLD [21]. When considering the role of liver in lipid metabolism, it is not surprising that liver cells are capable of storing lipids as energy reserves. However, hepatic lipid content is usually small (less than 5% of fat by wet weight); when the liver lipid reserves exceeds this percentage, it is assumed a liver steatosis condition [1]. In this regard, primary NAFLD is now considered as the hepatic manifestation of the metabolic syndrome [22-24], a cluster of disorders including central obesity, insulin resistance with or without type 2 diabetes mellitus, dyslipidemia and hypertension (see Chapter 7). The pathogenesis of NAFLD is attributed to a multi-hit process involving insulin resistance, oxidative stress, apoptotic pathways, and adipokines. In these days, there is no established treatment for this pathology except for weight loss and treating each component of the metabolic syndrome. Nevertheless, a large number of agents are being considered in clinical trials performed in patients with NASH. Hepatic steatosis should not be considered a benign feature, but rather a silent killer. This is supported by several studies demonstrating that this pathology has the potential to progress to NASH [25]. The pathophysiology of NAFLD is not fully elucidated, but the initial state involves accumulation of excess fat within the hepatocyte due to metabolic conditions leading to hepatic steatosis. The next stage involves the development of oxidative stress that causes structural damage to biomolecules, and activates inflammatory chain resulting in NASH [26]. Fat droplets accumulate in the cytoplasm of liver cells and their excessive accumulation may lead to cell damage or cell death, a phenomenon known as lipotoxicity [27, 28]. Lipotoxicity is associated with oxidative stress and the consequent activation of proinflammatory pathways, leading to immune infiltration, damage amplification, cell death and fibrosis [17].

2. Pathophysiology of Nonalcoholic Fatty Liver Disease and Steatohepatitis The pathogenesis of NASH is multifactorial. Among others, the causes of hepatocellular injury in steatotic liver include the following: oxidative stress, lipid peroxidation, mitochondrial dysfunction, and dysregulation of immune system. Because not all steatotic livers progress to NASH, some other environmental factors, or a combination of genetic factors, are thought to be required for progression to NASH and fibrosis. A number of factors aim to the multifactorial nature for the mechanism of NAFLD, including derangements in metabolic parameters, endotoxin-induced cytokine release and oxidative stress. The metabolic parameters include mitochondrial dysfunction, amino acid imbalance, hyperglycemia and imbalances in antiketogenic hormones in portal blood. Nonalcoholic fatty liver disease can progress to end stage liver disease; however, the cause of the NAFLD progression remains elusive. There is a hypothesis of “two hits” [29] postulating that liver fat accumulation per se is not harmful. The “first hit” is the excessive hepatic fat deposition. The “second hit”, for presumed transition of simple steatosis to NASH. Despite the involvement of many other proinflammatory mediators, lipid peroxidation is one

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hypothesis as the “second hit” to explain the development of the disorder in humans. However, the author of this hypothesis, in the light of more recent studies, proposed a modification of the two-hit model that places more emphasis on the role FFAs [30]. Accordingly, data from animal models suggest that oxidative stress contributes to steatohepatitis. Indeed, an increase of lipid peroxidation has been documented in humans as an important mechanism of progression of NAFLD [31]. The predominant metabolic features of the increased lipid peroxidation further suggest a close association between the oxidative imbalance and the dyslipidemia, thereby leading to functional deterioration of the steatotic liver [32]. There are no tested non-invasive diagnostic modalities to distinguish NAFLD and NASH, but new biomarker panels are approximating the liver biopsy in accuracy [4]. The diagnosis of hepatic steatosis and steatohepatitis or NASH is not yet possible without liver biopsy. Liver classical biochemical parameters, like aminotransferases, tend to be inconclusive and cannot be used reliably to confirm the presence or stage the extent of fibrosis. Nevertheless, a growing body of evidence strongly suggests that hepatic fatty acid amount may impact the degree of liver damage and therefore disease evolution [33]. Liver histology in NASH is usually classified through score systems based on morphological tissue parameters. Thus, Brunt et al. [34] proposed a classification of NASH as mild, moderate, or severe in relation to the degree alteration of parameters like steatosis, hepatocyte ballooning, lobular inflammation, and portal inflammation.

2.1. Hepatic Steatosis Hepatic steatosis is caused by an imbalance between the delivery of fat in the liver and its subsequent secretion or metabolism. Thus, fat accumulates when the delivery of fatty acids to the liver, either from the circulation or by de novo synthesis within the liver exceeds its capacity to metabolize the fat by β-oxidation or secrete it as very low-density lipoproteins (VLDL). A derangement in any of these pathways, alone or in combination, causes fat accumulation in the liver. Concerning the development of hepatic steatosis in NAFLD, it has been demonstrated that fat in the hepatocytes comes from multiple different origins including dietary lipids, fatty acids released from adipose tissue, and from de novo liver lipogenesis [1]. In humans it was demonstrated that after 2 weeks of high-fat diet (56% of energy as fat) lipid accumulation increased in hepatic tissue, while an isocaloric low fat diet (16% of total energy as fat) diminished liver fat content [35]. Moreover, studies in both humans and animal models indicate that adipose tissue is one of the major sources of the increased lipid movement to the liver [17]. Finally, together with dietary fat and fatty acids secreted from adipose tissue, hepatic tissue is also capable of de novo lipogenesis. Although the de novo lipid synthesis is probably not relevant in healthy lean subjects [36], it has been shown that in NAFLD patients it can generate up to 24% of liver fat [1]. In addition, fatty acids inside the hepatocytes are metabolized by one of two mechanisms: oxidation to generate energy or esterification to generate triglycerides, which are either integrated into VLDL particles for export or store within the hepatic tissue.

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Problems in any of these pathways could contribute to hepatic steatosis development [1, 37]. Defective mitochondrial/peroxisomal β-oxidation or microsomal cytochrome P450 4A ωoxidation and/or diminished ability of the liver to export lipids may be induced by mutations of genes encoding enzymes of lipid metabolism, as shown for mice deficient in liver carnitine acyl transferase-1 and triacylglycerol transferase with consequent impairment of fatty acid oxidation and triacylglycerol export, respectively. Alternatively, dietary fats can accomplish regulation of hepatic lipid metabolism through modification of gene transcription, as achieved with ω-3 and ω-6 long-chain polyunsaturated fatty acids (LCPUFAs) [38]. Under physiological conditions, LCPUFAs metabolism maintains an adequate ω-3/ω-6 proportion but any imbalance in this relation can influence metabolic-inflammatory pathologies including NAFLD [39].

2.2. Obesity and Insulin Resistance in Hepatic Steatosis Obesity promotion of steatosis is primary linked to the imbalance between the delivery of fat to the liver and its subsequent secretion or metabolism (positive energy balance). It is well known that high-fat diets foment an influx of ingested lipids to the liver promoting steatosis. In addition, high content digestible carbohydrates diets stimulate steatosis, due to the fact that permanent and excessive ingest of glucose elevates the amount of circulating insulin, the most lipogenic endocrine mediator. On the other hand, recent investigations have revealed that obesity plays a major role in the development of insulin resistance [40], known as a promoter of steatosis. Insulin resistance leads to steatosis in a similar way as high-digestible carbohydrates diets. Sustained hyperglycemia and/or hyperinsulinemia, promotes steatosis through the activation of lipogenic transcription factors (TFs). Nonetheless, de novo liver lipogenesis is up-regulated independently by glucose and insulin [41, 42]. Glucose acts directly by generating energy and acetil-CoA, a precursor of all de-novo synthesized lipids, and indirectly through the activation of TF carbohydrate response element binding protein (ChREBP). Insulin acts indirectly through the activation of TF sterol regulatory elementbinding protein-1c (SREBP-1c). Together, both TFs induce the synthesis of all the machinery involved in lipogenesis [43].

2.3. Fatty Acid Accumulation: Lipotoxicity It should be mentioned that steatosis may be a key step in the development of liver damage in NAFLD. Indeed, this metabolic derangement may affect the liver structure and function, but also other organs. These alterations may be caused by a phenomenon known as lipotoxicity, a condition that accounts for the pathologic changes in non-adipose tissues, such as the liver and pancreas, due to the adverse effects of excess fatty acid accumulation. In addition, lipotoxicity is associated to redox imbalance leading to increased formation of ROS [33]. Increased ROS production in presence of an excess of FFAs has been demonstrated in several models of NASH. For example, a research performed in CHO cells showed that cell

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death induced by saturated fatty acids depend on ROS production [44]. In addition, other studies have found that ROS plays a primary role in the activation stage of programmed cell death induced by FFAs [33]. Increased generation of ROS in the presence of large amounts of FFAs has been validated in many animal models of NASH [45, 46]. Livers of patients with NASH have augmented amounts of lipid peroxidation by-products, providing more evidence of increased ROS in this condition [37]. Oxidative stress may cause various kinds of functional and structural damage in non-immunological liver cells, like hepatocytes or endothelial cells [1]. In parallel, ROS stimulate the activation of other hepatic immune related cells, including Kupffer and stellate cells.

2.4. Free Fatty Acids as a Source of ROS in Hepatocytes Several experimental models have shown that excess of FFAs are a major source of ROS in NASH [45, 47, 48]. The most important ROS associated to this pathology are superoxide anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen molecules, establishing that oxidative stress exerts a central role in the development of NASH [6]. Increased hepatocyte FFAs triggers multiple processes capable of generating ROS [17]. First, FFAs could cause mitochondrial dysfunction, a process associated with enhanced ROS production and ATP depletion. Second, FFAs could generate an endoplasmic reticulum (ER) stress response, a phenomenon that stimulates ROS production. Third, increase in FFAs determines hyperactivation of some isofoms of microsomal cytochrome P450, leading to an increase in ROS formation. Fourth, a rise in cytoplasmic FFAs levels would increase ceramide formation, a well known pathway of ROS generation (figure 9-1). 2.4.1. Mitochondrial Dysfunction Mitochondria play a central role in cellular metabolism. It is the site of free fatty acid βoxidation and the citric acid cycle, which generates NADH and FADH2. The latter compounds transfer electrons to the respiratory chain, and finally to molecular oxygen, a process that generates ATP, the primary energy molecule of the cell. It has long been recognized that the mitochondrial electron transport chain is a main site of ROS generation [49]. Several lines of evidence show that NASH patients are characterized by abnormal mitochondria from both morphological and functional point of view [17, 37]. As previously mentioned, liver steatosis is at least in part, derived from an excessive hepatocyte lipogenesis, process that physiologically inhibit mitchondrial β-oxidation in order to avoid ATP depletion in a futile cycle of synthesis-oxidation of fatty acids. However, under steatosis conditions this mechanism could overload mitochondria with FFAs. Currently, it is well known that excessive accumulation of FFAs in liver cells directly induces mitochondrial dysfunction and oxidative stress. This process is associated with a sequence of events that includes mitochondrial depolarization, increased ROS production, release of cytochrome c, an electron carrier of the respiration chain between mitochondrial complexes II and III, and organelle dysfunction. FFAs-induced mitochondrial failure would be dependent on lysosomal disruption and activation of cathepsin B, peptidase involved in lysosomal metabolism, because the inhibition of these downstream events protects against FFAs-induced

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mitochondrial dysfunction and oxidative stress both in vitro and in vivo [50]. Reactive oxygen species production occurs because mitochondrial impairment causes an electron flow interruption that blocks respiratory chain leading to the transfer of electrons from respiratory intermediates to molecular oxygen to produce superoxide anions and hydrogen peroxide [45], which in turn starts a self-sustaining loop that worsen original mitochondrial injury [17]. This mitochondrial degeneration impairs β-oxidation capacity of the organelle, generating more free fatty acids accumulation, leading to a positive feedback mitochondrial dysfunction [37]. Additionally, mitochondrial injury may be a cause of reduced hepatocellular ATP stores in NASH [10], hampering the regeneration of antioxidant defenses, the reparation of damaged organelles and the maintenance of cellular homeostasis, thus encouraging the development of oxidative stress. Finally, ROS-associated release of mitochondrial cytochrome c is intimately linked to mitochondrial dependent apoptosis process related to caspase cascades activation.

Figure 9-1. A comprehensive model detailing the different sources of hepatocyte free fatty acids (FFAs) and their implication on non alcoholic fatty liver disease (NASH) development related to oxidative stress generation via different pathways. Dual arrows imply bidirectional relation.

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2.4.2. Endoplasmic Reticulum Stress Liver ER is a central organelle for lipid metabolism, biological membranes assembly and lipoprotein management [51], constituting a major site of FFAs accumulation. Since this organelle comprises more than half of the total membranes in the hepatocyte and has the highest cellular calcium concentration, together with a unique oxidative environment to support disulfide bond formation [33], it becomes in the best cellular place for FFAs spontaneous precipitation and oxidation. This process is associated with deficiencies in protein synthesis and folding. This phenomenon is related with the disruption of the organelle, called ER stress [33]. The RE stress is highly related with ROS generation [52]. Maintenance of integrity of ER membrane structure is essential to physiological oxidation of proteins, associated to disulfide formation and cellular Ca2+ handling [53]. Hepatic steatosis and NASH are associated with known triggers of the ER stress. Accumulation of saturated FFAs and its calcium precipitates in ER decrease the fluidity of the organelle, affecting their ability to fold and export proteins. Accumulation of unfolded protein initiates the activation of an adaptive signaling cascade known as unfolded protein response (UPR) [54]. Unfolded protein response could derivate in ROS generation by extrinsic and an intrinsic pathways. In the first case, unfolded protein accumulation in the ER may elicit Ca2+ leak into the cytosol. The very close proximity of ER and mitochondria leads to accumulation of this cation near mitochondria allowing it to enter to this organelle and depolarize its inner membrane. This process triggers increased mitochondrial ROS production, cytochrome c release and mitochondrial dysfunction. High levels of ROS generation within the mitochondria further increase ER disruption and sensitize the Ca2+ release channels at the ER membrane, by oxidizing a critical thiol in the ryanodine receptor causing its inactivation, both processes enhancing Ca2+ release from ER create a vicious cycle that threaten to cell survival [52]. In addition, this vicious cycle could activate both mitochondria-dependent and mitochondria-independent caspase cascades, the latter associated to protein kinases Ca+2 dependent (PCKs). The intrinsic pathways of ROS generation by ER is related with oxidative protein folding, a highly regulated process catalyzed by a family of ER oxidoreductases, including protein disulfide isomerase. In these reactions, molecular oxygen serves as the terminal electron acceptor for disulfide bond formation. The enzymes ER oxidoreductases use a flavin-dependent reaction to pass electrons directly to molecular oxygen, a reaction that has the potential to generate ROS. It has been estimated that near 25% of the ROS generated in secretory specialized cells may result from the formation of disulfide bonds in the ER during oxidative protein folding, ROS that otherwise would be removed by antioxioxidant defenses [54]. However under ER stress this ROS production is enhanced because the activation of UPR allows only properly folded proteins to exit the ER. Misfolded proteins are either retained within the ER lumen in complex with molecular chaperones to repair them. Catalyzed reparation of these proteins use reduced glutathione (GSH), one of the most important endogenous antioxidant, to return thiols involved in non-native disulfide bonds to their reduced form so they may again interact with ER reductase to be reoxidized. This process generates a futile cycle of disulfide bond formation and breakage, in which each cycle would generate more ROS and consume GSH. Alternatively, because both protein folding and unfolding are highly energy-dependent processes, ATP depletion as a

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consequence of protein misfolding, could aggravate oxidative stress [52]. Recent studies show the presence of ER stress in mice with hepatic steatosis. In addition, a new small clinical trial has demonstrated that there is a progressive degree of activation of the UPR between steatosis toward steatohepatitis in NAFLD patients [54]. 2.4.3. Cytochrome P450 Activation Cytochromes P450 are an integral ER superfamily of heme-monoxygenases essential to metabolize an important range of compounds for a proper elimination from the body. While a limited number of these enzymes are involved in the synthesis of steroid and bile acid production, most cytochromes P450s metabolize xenobiotics or foreign compounds including several drugs, toxic compounds and chemical carcinogens. Cytochromes P450 catalyze the oxidation of carbon and nitrogen functional groups usually resulting in the incorporation of an alcohol moiety. In some cases, an epoxide is incorporated to an aromatic ring that is spontaneously converted to an alcohol by non-enzymatic reaction with water, or transformed to a trans dihydrodiol by epoxide hydrolases [21]. Paradoxically, some of these enzymes also can cause cell toxicity by different pathways, among other, the generation of ROS through the oxidation of several compounds [17]. Cytochromes P450 that metabolically activate toxic compounds and carcinogens are limited to a few isoforms including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2E1, and in a lesser extent the CYP3A subfamily. Cytochromes P4501A1 and CYP1B1 mainly activate polycyclic aromatic hydrocarbons. Cytochrome P4501A2 carries out the N-oxidation of heterocyclic amine food mutagens and arylamines and finally CYP2E1 metabolizes numerous compounds, such as aromatic, aliphatic and halogenated hydrocarbons, many of which are industrial solvents, and some of which are cancer suspect agents. The increased availability of FFAs determines the activation of microsomal cytochrome P450 isoforms CYP4A10/4A14 and especially CYP2E1, both involved primarily in FFAs ω-oxidation, leading to an increased ROS production [17]. The mechanism of this reaction involves the flavoprotein-mediated donation of electrons and during the catalytic cycle, P450s use hydrogen from NADPH to reduce O2 leading to the production of H2O2 and superoxide radical [55]. Interestingly, even in the absence of substrate, these enzymes can generate ROS. This phenomenon, known as futile cycling, is due to P450-derived NADPH oxidase activity, a process independent of exogenous xenobiotic substrates [56]. These ROS generated within the ER membranes produce peroxidation of LCPUFA, a process that foment oxidative stress and ER stress. On the other hand, FFAs indirectly could cause induction of P450s ωoxidation, because in the presence of lipid excess, increased FFAs oxidation leads to an increase in ketone bodies, mediators that are inducers of these enzymes [21]. The participation of P450s ω-oxidation in NASH oxidative stress was proven by a study in a genetic model of GSH synthesis deficiency in mice, NASH spontaneously develops and is associated with elevated CYP2E1 expression and increased lipid peroxidation that is decreased by addition of the CYP2E1 inhibitor diallyl sulfide [21]. In animal models of high fat diet-NASH induced, several studies have demonstrated an elevated expression and activity of CYP2E1. Obese humans have both elevated CYP2E1 activity and induction, indicating that CYP2E1 is related to hepatic pathology resulting from morbid obesity. Moreover, the level of P450 correlated well with the degree of steatosis. Likewise, in rodents

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a similar high level of CYP2E1 activity was found in non-diabetic patients with NASH, indicating that the activity of this P450 isoform is elevated in humans with this pathology. Also, CYP2E1 protein levels are spontaneously induced in patients with NASH [21]. 2.4.4. Ceramide Formation Ceramide is a sphingolipid, a category of lipids derived from the aliphatic amino alcohol sphingosine. Sphingolipids are important structural constituents of biological membranes in animal cells, mostly plasma membranes. During the last years, sphingolipids have been studied due to their role in signal transduction, proliferation, differentiation and immune response [49]. Despite its physiological role, a growing body of researches also suggests that ceramide and/or its derivatives can induce cellular ROS production in different cell models through the activation of NADPH oxidase [57] and inducible nitric oxide synthase (iNOS) [58]. Also, ceramide might down-regulate antioxidant enzymes [59], and/or alter the mitochondrial function [60]. In endothelial cells ceramide was able to induce endothelial dysfunction via superoxide generation, resulting in peroxynitrite formation. This activation was blocked by NADPH oxidase specific inhibitors, N-vanillylnonanamide, apocynin, and diphenylene iodonium, but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes [57]. On the other hand, in different cell models ceramide can induce apoptosis by activating the pro-inflamatory TF nuclear factor kappa B (NF-қB) [61], which upregulates the expression of iNOS. The increased formation of nitric oxide (NO) induce the production of reactive nitrogen species (RNS), specially peroxynitrite, a powerful long life oxidant agent, which is probably an important cause of apoptosis [62]. Different iNOS inhibitors, such as nicotinamide and aminoguanidine, diminished NO formation under these conditions in vitro and prevent ceramide β-cell apoptosis and diabetes in vivo [63]. In relation to antioxidant enzymes, ceramide was reported to participate in the regulation of mitochondrial manganese dependent superoxide dismutase (Mn-SOD) and catalase in various cell types. Ceramide mediated the generation of ROS and subsequent activation of redoxsensitive transcription; these factors may be involved in the up-regulation of the Mn-SOD gene expression. The mechanism for ceramide-induced inhibition of catalase is not clear, but the inhibitory effect of ceramide on phosphatidylinositol-3-kinase has been reported to be involved in this process [45]. Finally, naturally occurring 16 carbon ceramide was shown to cause an increase in ROS generation through mitochondria, promoting cytochrome c release [60]. The de novo ceramide production or its glycosyl derivatives, has been proposed as an important mediator in the cytotoxicity of saturated FFAs in NASH [64]. For example it was known that FFAs, induce ceramide synthesis in different cell types, and inhibitors of de novo ceramide synthesis diminished the induction of apoptosis by FFAs. The role of ceramide toxicity in liver diseases has been confirmed in several model systems especially in mouse with truncated ceramide synthesis. These animals were more resistant to alcohol-induced cirrhosis than normal mice. In addition, hepatocytes from the transgenic mice were less sensitive to TNF-α induced apoptosis [65].

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2.5. Non FFAs Oxidant Species Production In addition to the FFAs associated generation of ROS, some NASH patients could have type II diabetes or insulin resistance. Both conditions can expose the liver to exacerbated levels of glucose, process that could lead to glucotoxicity. Despite glucotoxicity is poorly studied in liver models, it is a well known source of ROS in other cellular models, such as β-cell [53] and neurons [66]. The glucose-induced rise in ROS would be associated to different mechanism, for example mitochondrial dysfunction and ER stress, induction of lipogenesis, activation of immune system and stimulation of fibrosis by the route of advanced glycation end-products (see Chapter 8) [17, 53, 67]. On the other hand, beyond the ROS production by processes directly associated to liver hepatocyte metabolism, there exists a primary hepatocyte ROS production derived to extrahepatic nonimmunological tissues, especially adipose tissue. This would be a second line of association between obesity and NASH, because several lines of evidence suggest that adipose tissue generates and releases a variety of inflammatory molecules, such as TNF-α [1], able to stimulate ROS production in many non immunological cell types including hepatocytes [40]. Moreover, plasma TNF-α level correlates with body fat mass [10].

2.6. Liver Oxidative Stress Despite the fact that the presence of elevated levels of ROS could be dangerous for any kind of cells, the hepatocyte is one of the best equipped for this type of aggression, because unlike other cells, multiple liver physiological processes are associated with the generation of large amounts of oxidant species. For example, xenobiotic detoxification, permanent fatty acids oxidation, protein secretion and peroxisomal function, constitute challenges forcing the hepatocyte to develop the best antioxidant defenses of mammalian cells, enabling the liver to survive adequately with ROS concentrations being toxic for other cells. Hence under normal conditions, hepatic aerobic metabolism involves a steady-state production of pro-oxidants, such as ROS and RNS, which are balanced by a similar rate of their consumption by antioxidants. However, as mentioned above, the steatotic liver seems to be particularly susceptible to oxidative damage. Oxidative stress occurs when there is an imbalance between the generation of ROS and the antioxidant defense systems in the body so that the latter become overwhelmed [68]. Oxidative stress is developed in NAFLD at the stage of steatosis and is exacerbated in steatohepatitis. This metabolic derangement is accompanied by a decrease in both glutathione content and the activity of antioxidant enzymes, associated with liver cytochrome P450 CYP2E1 isoform induction. Additionally, the antioxidant capacity of plasma is inversely correlated with the progression of NAFLD toward NASH. Recently, it was suggested an impaired glutathione metabolism towards an oxidant status in NASH patients, correlating this with a higher intake of saturated fat and a lower intake of carbohydrates [69]. Finally, a recent study in a rat model it was shown that shows that increased activity of the renin-angiotensin system through the vasoconstrictor Angiotensin II causes development and progression of NAFLD by increasing hepatic ROS [70]. Oxidative stress generates the

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chemical alteration of essential biomolecules causing their inactivation; hence it leads to direct hepatocyte cell injury. In addition, oxidative stress by-products, such as some toxic aldehydes, have longer half-lives than ROS and are able to damage the hepatocyte and diffuse out to extracellular targets [71]. The well established major oxidative stress occurrence in NASH pathogenesis was demonstrated in human biopsies by several markers [72]. First, there is a significant increase of lipid peroxidation by-products (e.g. thiobarbituric acid reactive substances, TBARS; malondialdehyde; 4-hydroxynonenal) [73, 74]. Second, it has been found an increase of nitrotyrosine protein modifications [75]. Third, it has been detected the presence of DNA hydroxylation [73]. Fourth, there are increased plasma oxidative stress parameters such as thioredoxin, among others [76]. Fifth, reduction of antioxidant defenses, including diminished levels of GSH and coenzyme Q10 and lower antioxidant enzymes activities such as glutathione S-transferase, CuZn-superoxide dismutase and catalase [74]. The combination of direct oxidative stress effects and their by-products effects have the intrinsic ability to generate cell necrosis. However, before this irreversible process occurs, oxidative stress generates an adaptive response, aimed to avoid damage propagation and stimulate regeneration: this is the inflammation process. Oxidative stress derivate inflammation is a finely regulated process that involves several changes in genetic expression and important modifications in hepatocyte metabolism. Many of these changes would be target to trigger apoptosis, probably to avoid a malignant transformation associated to a chronic and moderate oxidative stress or to prevent the explosive inflammation response associated to cell necrosis.

2.7. Liver Inflammation Inflammation is a complex, conserved and non-specific biological response of cells and tissues to injurious stimuli, such as cell necrosis, exposition to pathogens and toxic substances, among others. It is a protective biological response that involves the local vascular system, the immune system, and various cells within the injured tissue in order to remove the harmful as well as initiate the reparation of the affected tissue. Inflammation is characterized by vasodilation and augment in vascular permeability with the subsequent increased movement of plasma and immune cells, specially macrophages, neutrophils and monocytes, from the blood to the affected tissues, process called infiltration. This phenomenon begins with a cascade of biochemical events related with the activation and release of inflammatory mediators, including cytokines and prostaglandins, by resident immune and non-immune cells of the injured tissue. Inflammation mediated by oxidative stress is originated by two main pathways; the intrinsic and the extrinsic pathways. The intrinsic pathway relates to the ability of ROS to induce directly in the affected parenchymal cells the expression of different kinds of proinflammatory mediators, without immune cell mediation. For example, ROS can promote directly the formation and release of inflammatory mediators in parenchymal cells by regulated molecular mechanism independent from external stimulus. The extrinsic pathway is associated with the ability of parenchymal oxidative stress to induce the release of inflammatory mediators by surrounding resident immune cells. This

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process is mediated mainly by the ability of ROS to trigger apoptosis in an affected cell, through a controlled series of events related with the sequential activation of several proteins [67]. Apoptotic cell bodies have the ability to induce the activation of different immune cells and stimulate the differentiation of pro-immune cells to their functional state, both capable to express large amounts of new proinflammatory mediators. These mediators induce the relaxation of endothelial cells junctions and stimulate the expression of adhesion molecules in non-immune cells of the injured tissue. Both of these processes are responsible for the influx of circulating not resident immune cells into the affected tissue [37]. This infiltration, carried out by cells of innate immune system, is the first organism defense barrier to contain an aggression. Indeed, it plays and important role in the development of a later adaptive immune response, if necessary, and it is fundamental to the regeneration of the damage tissue. On the contrary, chronic inflammation is a pathological condition that favors cell death and threatens to tissue reparation because inflammatory mediators and infiltration stimulate cell death directly and indirectly. Then, their maintenance in time prevents cellular regeneration and begins to affect the healthy surrounding tissue. During inflammation, one of the most important molecular mechanisms to stimulate cell death is the generation and release of powerful oxidant species such as ROS and RNS to affected tissue. These species have the ability to destroy pathogens and infected or modified cells that in most cases are more susceptible to an oxidative threat than healthy cells. Despite of its recognized efficiency, this mechanism has a limitation; when the oxidant species do not meet its goal within a reasonable period they begin to affect the healthy surrounding cells spreading oxidative stress conditions, which in turn induces inflammation, generating a vicious cycle that propagates and matures the original inflammatory response, being a key step in chronic inflammation [77]. Chronically inflamed hepatic tissue is the most important classical marker of NASH and practically defines the disease, being the marker that makes the difference between simple steatosis and steatohepatitis. Unlike other diseases such as viral hepatitis and or infection, in NASH the main injurious stimuli that induce the inflammation is the hepatocyte oxidative stress, phenomenon that starts the vicious circle that in fatty liver conditions is very difficult to be stopped. As mentioned below, virtually in all pathologies related with oxidative stress, especially in NASH, oxidant species induce directly the expression of pro-inflammatory mediators. This process occurs through the activation of pro-inflammatory TFs such as NF-κB and activating protein-1 (AP-1), among others, in all cell types of the affected tissue. These TFs are sensitive to a decrease in intracellular redox potential [43]. Nuclear factor kappa-B preferentially induces the transcription of pro-inflammatory mediators including paracrine molecules such as TNF-α and interleukin-1, cell surface molecules such as TNF-α receptor and intracellular molecules like iNOS. In contrast, AP-1 typically stimulates the expression of pro-apoptotic proteins (e.g. p53) and adhesion molecules. Hepatocyte released paracrine molecules react with surrounding endothelial cells stimulating them to express other proinflammatory mediators, especially adhesion molecules. In parallel, these paracrine molecules activates surrounding immune resident cells that starting to release large amounts of ROS and RNS and secrete more inflammatory mediators.

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2.7.1. Liver Endothelial Cells Liver sinusoidal endothelial cells constitute the sinusoidal wall, also recognized as the endothelium, or endothelial lining. The endothelial cells can be regarded as unique capillary cells which differ from other tissue capillary cells, because of the presence of several open pores or fenestrae that lacks the classical diaphragm and a basal lamina underneath the endothelium. Fenestrae filter all the fluids, solutes and particles that are exchanged between the sinusoidal lumen and parenchymal tissue, allowing only particles smaller than the fenestrae to move in a bidirectional way [78]. Injured sinusoidal endothelial cells play direct and indirect roles in the development of inflammation and propagation of the initial hepatocyte oxidative stress. During these processes, there are changes in the molecular expression profile of the normal hepatic endothelial cells. These changes are characterized by expression of high levels of inflammatory molecules such as MIP-1β (Macrophage inflammatory protein 1β), and ITAC (IFN-gamma-inducible T cell alpha chemoattractant) and adhesion molecules such as ICAM-1 (inter-cellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1). In addition, these cells begin to directly generate ROS by enzymatic and not enzymatic mechanism [79]. 2.7.2. Kupffer Cells The most important of the liver immune cells are Kupffer cells, the resident liver macrophages, which play significant roles in immunomodulation, phagocytosis, and biochemical attack [77]. Any liver insult, including inflammatory substances derivates from hepatocytes by oxidative stress, immediately triggers Kupffer cells activation [80]. The presence of an excess of lipids, including neutral ones, such as triacylglycerols (TGs), appear to sensitize these macrophages to liver insults, for example, adding TGs to lipopolysaccharide enhanced the ability of the latter to induce the expression of proinflammatory mediators in Kupffer cells. As Kupffer cells account for 70-80% of the whole body macrophages [80] its chronic activation results in the secretion of large amounts of RNS, derived from the induction of iNOS, [77]. This process increases tremendously the original liver oxidative stress, becoming in a key step in the development and progression of NASH. In addition, activated Kupffer cells are known to generate and secrete important quantities of inflammatory mediators associated to liver injury, such as TNF-α, interferon α and β (INF α/β), interleukin-1β, interleukin-6 and granulocyte colony-stimulating factor (GCSF) [81-83], enhancing the original inflammatory response. This also directly affects hepatocytes. For example, TNF-α, one of the most powerful pro-inflammatory cytokines that are the most abundant cytokine in NASH, has the ability to generate oxidative stress in healthy hepatocytes promoting lipolysis, increasing cytosolic FFAs and interrupting mitochondrial electron transport chain with similar effects than FFAs, ROS and Ca2+, fomenting apoptosis and/or necrosis [17]. 2.7.3. Infiltration The joint activation of hepatic resident immune and endothelial cells amplifies the original inflammation signals, triggering immune infiltration with different types of circulating inflammatory cells. Immune infiltration is associated with the recruitment of circulating macrophages and leukocytes, mostly monocytes and neutrophils, into the liver

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vasculature [84, 85]. In NASH, the major function of these cells is to remove dead cells and cell debris in preparation for tissue regeneration. However, because of the nature of these immune cells, during long-term infiltration non-affected neighbor hepatic tissue may also be damaged, which can aggravate the original liver injury [86]. Activation of infiltrated immune cells release more ROS and RNS, thus further enhancing oxidative stress. In addition, if the rate of removal of dead cells and regeneration of tissue is lesser than the parenchymal destruction rate, such as in NASH, infiltration cells become more active and begin to secrete the same inflammatory paracrine molecules secreted by Kupffer cells. This event amplifies the damage to its highest level fomenting the irreversible loss of the affected parenchyma which is replaced by non functional tissue in the process called fibrosis.

2.8. Fibrosis Fibrosis is the replacement of the parenchyma tissue by fibrous connective tissue as a reparative or reactive process. It is a regenerative pathological response that involves the synthesis, release and deposit of an special kind of extracellular matrix characterized by their density and rigidity. This dense connective matrix, also called dense fibrous tissue, has collagen fibers as main element, composed of type I and type III collagen that forms strong structures which separates affected tissue from different and/or healthy tissue. Collagen rich connective tissues are manufactured by fiber-forming cells or fibroblasts in response to a complex network of signals associated to final stages of pathological states, including heavy oxidant species, pro-inflammatory mediators and apoptotic bodies, among others. Obesity and related insulin resistance promotes fibrosis progression by different mechanisms including steatosis, hyperleptinemia, increased TNF-α production and impaired expression of peroxisome proliferator-activated receptors-γ receptors (PPAR-γ) [87]. The renin-angiotensin system, plays a vital role in blood pressure regulation (see Chapter 2), and appears to promote hepatic fibrogenesis too [70]. Fibrosis is one of the final stages of all chronic liver disease; it is carried out by activated fibroblast derived from hepatic stellate cells. 2.8.1. Hepatic Stellate Cells Hepatic stellate cells (HSTs) are pericytes located in the perisinusoidal space of the liver. As a relatively undifferentiated cell, under physiological conditions it usually remains in a quiescent state, but still has the ability to differentiate into fibroblast, macrophages or contractile myofibroblasts as required [88]. In response to chronic inflammation and oxidant species these cells experience a massive activation and become highly proliferative and fibrogenic beginning the generation and deposition of extracellular matrix components, mainly type I collagen [80]. Hepatic injury in NASH related with oxidative stress, and inflammation leads to cytotoxic events that activate hepatic stellate cells and deposition of collagen [89]. Activation of stellate cells is the key step in the development of liver fibrosis. This process is mediated mainly by ROS, RNS and inflammatory cytokines derived from damaged hepatocytes and activated Kupffer cells, but mainly from activated infiltrating immune cells [80]. Injured sinusoidal endothelial cells can also play an important role in hepatic fibrosis participating in both early HSTs proliferation and plasmin activation [90].

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These processes are associated with fibrosis [91]. Stellate cells also can secrete ROS and other inflammatory molecules [17, 80] favoring the perpetuation of the inflammatory response. Hence, these cells have the ability to directly induce inflammation, hepatocyte necrosis, and liver fibrosis, all of the classical histological markers of NASH [37]. 2.8.2. Fibrogenesis in NASH Profibrogenic mechanisms operating in NASH are partly in common with those observed in other chronic liver diseases. The differences are related with the presence of increased circulating adipokine levels, altered glucose metabolism and the hormonal profile associated with the metabolic syndrome. These conditions might have a specific role in the induction of fibrogenesis in NASH. Reactive oxygen species and RNS stimulate the progression of liver fibrosis and increase the procollagen expression in HSTs. Oxidative stress-derived molecules, for example reactive aldehydes, induces activation of these cells. Pro-inflammatory mediators stimulate immune cell release of fibrogenic factors, such as platelet-derived growth factor and transforming growth factor β1, which stimulate the profibrogenic actions of HSTs [17]. Hepatocytes apoptotic bodies stimulate fibrogenesis in vivo [92] because phagocytosis of these particles by hepatic stellate cells activates these cells and stimulate NADPH oxidasemediated production of ROS. Angiotensin II induces profibrogenic actions in HSTs, which express all components of the renin–angiotensin system, moreover interference with this system attenuates fibrosis development in different laboratory models of chronic liver disease [93]. In relation to comorbidities, insulin resistance is a potent predictor of fibrosis, because elevated glucose or insulin levels up-regulate transforming growth factor β (TGF-β) and connective tissue growth factor. Hyperglycemia could also affect hepatic stellate cells biology because receptors for advanced glycation end-products are expressed in these cells and have a role in migration of activated hepatic stellate cells [94]. Leptin, the most abundant adipokine in obesity conditions is a potent profibrogenic factor, acting directly on hepatic stellate cells [95]. 2.8.3. Cirrhosis The final stage of liver fibrosis is cirrhosis, condition defined as huge and extended distortion of normal hepatic architecture. Cirrhosis is characterized by replacement of liver tissue with regenerative nodules surrounded by dense layer of fibrotic tissue. As time goes by regenerative nodules develop in the midst of scars which may result in advancement and neoplastic transformation of the cirrhosis. Hepatic cirrhosis is usually considered irreversible and treatment is only supportive. The evolution rate of early NASH fibrosis to cirrhosis and the morphology of the last condition vary from person to person, presumably, because the wide range of comorbid conditions that could accompany NAFLD during their progression and the different individual’s response to those pathologies.

3. Antioxidant Therapies Up to date, there is no an effective medical treatment available for NASH patients. Therapy is predominantly aimed at controlling comorbid pathologies, such as obesity, insulin resistance, and dyslipidemias [96]. Weight loss is mostly advocated but it is difficult to

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achieve in the majority of patients. Indeed, some affected individuals have been reported to worse their hepatic damage with excessive weight loss [97, 98]. Table 9-1. Antioxidant tested for the treatment of NASH Agents Glycyrrhizin

Vitamin E

Proposed mechanisms Free radical scavenging Inhibition of FFAs accumulation Anti-inflammatory Immunomodutation Free radical scavenging Regeneration of oxidized glutathione to the reduced active form.

Probucol

Free radical scavenging Promotes endogenous antioxidants synthesis

N-acetylcysteine

Free radical scavenging Stimulates glutathione synthesis

Vitamin C

Sadenosylmethionine

Free radical scavenging Regenerates oxidized vitamin E and glutathione to the reduced active forms. Glutathione synthesis through formation of cysteine

Betaine

Increasing S- adenosylmethionine

Genistein

Free radical scavenging Anti-inflammatory Antifibrotic

References Wu et al. 2008 [100]

Lavine 2000 [106] * Hasegawa et al. 2001[107] * Kugelmas et al. 2003 [108]* Vajro et al. 2004 [109]* Harrison et al. 2003 [110]* Sanyal et al 2004. [7]* Dufour et al. 2006 [6]* Merat et al. 2002 [115]* Merat et al. 2002 [117]* Merat et al. 2008 [108]* Tokushige et al. 2007 [118]* Thong-Ngam [119] Baumgardner et al. [120] Gulbahar et al [121] * Pamuk and Sonsuz [122]* De Oliveira et al. [123]* Shireen et al. 2008 [127] Harrison et al. 2003 [110]* Vendemiale et al. 1989 [135] † Labo and Gasbarrini 1975 [136] † Frezza et al 1990, [137] † Mato et al 1999 [138] † Miglio et al [142]* Abdelmalek et al. [143]* Yalniz et al. 2007 [145]

*Clinical Trials † Tested in no NASH liver disease models.

In relation to liver steatosis, a major concern in treating is to prevent progression to NASH, but there is not general consensus on the effectiveness of any therapeutic agent for treating fatty liver [25]. However, the role of oxidant processes in the progression of hepatic steatosis to NASH and in NASH worsening, opens the door to an intervention pathway, being logical to think that antioxidant therapies oriented to the neutralization of oxidative stress, such as inhibition of oxidant species production and scavenging of oxidant species would be an effective therapy in treating NASH by amelioration of the pathology driving forces and reduction of the secondary effects [11] (table 9-1). 3.1. Treatment of Conditions that Induces the Liver Oxidative Stress

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Stopping or reducing liver metabolic oxidant species production would seem to be the best strategy to treat NASH. In fact currently most accepted treatments are based on this strategy, such as weight loss, hypolipemiant and hypoglycemiant therapies. The first two strategies are focused in lipotoxicity reduction, process highly associated to oxidative stress. In addition, weight loss would limit oxidant species generation through the reduction of adipose tissue, an important source of inflammatory molecules associated to NASH development. Hypoglycemic agents, associated to insulin resistance therapy, would tend to reduce liver glucose levels, and FFAs lipogenesis, both processes also associated to liver oxidative stress. Recently, the utilization of substances that directly prevent lipotoxicity by inhibition of FFAs accumulation, such as vegetable bioactive compounds (e.g. glycyrrhizin) is beginning to be studied. 3.1.1. Metabolic Treatments Several clinical trials have studied the effects of weight loss in NASH using different strategies such as diet, exercise, and gastric surgery. Most of that trials show improvements in NASH markers [10], including histological parameters. Antidiabetic therapy studies with drugs (e.g. metformin and glitazones) also have shown damage reduction. On the contrary, antihyperlipidemic therapy using diverse pharmacological agents, such as fibrates and statins has not been conclusive. In spite of the absence of absolute evidence of the benefits of these therapies, metabolic control of oxidant species production seems to be a good alternative to treat NASH. Preventing the production of new oxidant species by the original metabolic inductors could be expected to have benefits in these patients. However, it should also be considered that the presence of large amounts of already formed oxidant molecules could catalyze their own propagation, thereby increasing cell damage, what should need a specific pharmacological therapy. Moreover, this metabolic prevention of oxidant species production probably would not finish in short term with the heavy immune ROS and RNS generation associated to immune activation. Hence, the direct elimination of produced oxidant species is still very important. 3.1.2. Glycyrrhizin Glycyrrhizin is a triterpene glycoside considered the major bioactive compound of licorice root extract. Licorice is one of the most traditional medicinal plants; it has an ancestral use in traditional Chinese medicine for the treatment of several inflammatory diseases [99]. Glycyrrhizin has a variety of pharmacological properties including antiinflammatory, antioxidant, and immune-modulating activities and has been used to reduce liver inflammation and hepatic injury. The mechanism by which glycyrrhizin improve NASH has been studied in HepG2 (human liver cell line) and might be associated with the prevention of FFAs-induced toxicity and subsequent cell apoptosis [100]. These effects are mediated by 18 beta-glycyrrhetinic acid (GA), the biologically active metabolite of glycyrrhizin. This metabolite also stabilizes lysosomal membranes, inhibits expression and activity of the proapoptotic lysosomal peptidase cathepsin B, reduces FFAs-induced oxidative stress and inhibits mitochondrial cytochrome c release. Animal models of high fat diet-induced NASH show that GA prevents hepatic lipotoxicity and liver injury in vivo [100].

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One of the most remarkable aspects of glycyrrhizin is that in addition to their direct antioxidant properties, it also has the ability to attack the cause of steatotic hepatocyte oxidative stress: the lipotoxicity. This feature makes the glycyrrhizin a promising agent to complement a traditional antioxidant therapy that is focused only in remove, in a directly or indirectly way, previously generated oxidant species. Moreover, as licorice root extract has an ancestral use in the treatment of liver diseases, glycyrrhizin is in a privileged position to be proved in NASH clinical trials.

3.2. Oxidant Species Scavengers As oxidant species play a major role in NASH pathogenesis, antioxidant supplementation has been thought as a therapeutic option [25], and many antioxidants have been studied in NASH [101]. An antioxidant agent is any molecule that favors, through any mechanism, the loss of reactivity in oxidant molecules. Direct antioxidants avoid oxidative reactions at any level and indirect antioxidants stimulate the generation and/or activity of direct antioxidants. In relation to direct antioxidants, vitamins E, probucol and vitamin C appear to be the only tested in clinical trials, and related to indirect antioxidants only N-acetylcysteine, Sadenosylmethionine and Betaine have been studied. Recently, other antioxidant substances, such as genistein, a phytoestrogen found in soybeans, shown promising in animal models of the pathology; however there are no clinical trials that prove its effectiveness in human patients. 3.2.1. Vitamin E The term vitamin E is related to a family of lipid soluble substances called tocopherols and tocotrienols, each with different suffix; α, β, γ, and δ. The α-tocopherol is the most abundant natural form and is the major tocopherol in human plasma [71]. This vitamin has powerful free radical scavenging abilities, breaking free radical chain reactions, that is of principal importance in lipid peroxidation due to interrupt unsaturated fatty acids degradation [6]. Animal models of liver disease associated with oxidative damage have shown that vitamin E reduces lipid peroxidation, corrects oxidative stress and improve fibrosis [102105]. For example, in a mouse model of NASH, the intervention group significantly improved reduced glutathione reserves, with a subsequent decline in levels of TBARS and improvement in fibrosis histological score [65]. In clinical trials vitamin E has been studied alone or associated with other therapeutic agents. In the first study, Lavine [106] used increasing doses of vitamin E, up to 1200 IU/day, in a small group of affected children demonstrating improvement in aminotransferases, but liver histology was not analyzed. In the second study, Hasegawa et al. [107] in a little uncontrolled pilot trial, demonstrated fibrosis improvement in 66% of adult NASH patients treated with vitamin E in doses of 300 mg/day during 12 months. Later, in other pilot study carried out by Kugelmas et al. [108] with NASH adult patients, vitamin E and aerobic physical activity supplementation showed no effects after 3 months of 800 IU/day vitamin supplementation, suggesting that in short-term interventions this vitamin provides no apparent added benefit or physical activity masked possible vitamin effects. However, Vajro

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et al. [109], in a pediatric randomized placebo-controlled trial, showed hepatic enzyme improvement in 14 children with hepatic steatosis treated during 5 months with low doses of vitamin E (400 IU daily for the first 2 months and 100 IU daily for the final 3 months). Interestingly, the subgroup of treated patients that did not show weight loss during the trial, showed improvement in aminotransferases levels, suggesting that vitamin E therapy may be applicable in obese children who are unable to adhere to a calorie restriction treatment. Moreover, the most complete randomized placebo-controlled clinical study of vitamin E carried out by Harrison et al. [110], in adult patients with biopsy-proven NASH, showed a significant improvement in fibrosis score of liver biopsies in the intervention group after 6 months. However, in this group patients received 1,000 daily UI of vitamin C, in addition to 1,000 IU daily of vitamin E. Finally, other currently published vitamin E intervention trials evaluate the effect of vitamin E in combination with other therapeutic products not necessarily antioxidants agents. Sanyal et al. [7] showed in a 6 months pilot study that mixed therapy with vitamin E and pioglitazone (a hypoglycemiant drug) generates a greater improvement in NASH histology than only vitamin E therapy. Nonalcoholic steatohepatitis patients were randomized with 400 IU daily of vitamin E alone versus vitamin E plus 30 mg daily of pioglitazone. Both intervention groups show improvement in liver histology and aminotransferases normalization. However, the mixed therapy group showed greater improvement in several biopsy parameters such as steatosis, inflammation, hepatocyte ballooning, and Mallory hyaline at the end of the trial. In other more recent randomized placebo-controlled trial, Dufour et al. [6] demonstrated NASH improvements with a vitamin E-based therapy plus ursodeoxycholic acid (UDCA), a secondary bile acid with well known hepatic cytoprotective properties and immunomodulatory effects. Initial biopsy-proven NASH patients were randomly assigned to receive doses of UDCA 12–15 mg/(kg/day) with 400 IU of vitamin E twice a day, UDCA with placebo, or placebo with placebo during 2 years before a second biopsy. The initial inter-group body mass index did not change significantly during the 2 years. However, the UDCA plus vitamin E group improved laboratory values and hepatic steatosis of NASH patients. Interestingly, the UDCA alone group had no differences in any parameter with the placebo-placebo group. 3.2.2. Probucol Probucol is a synthetic polyphenol drug, with hypolipidemiant effects, widely used in heart and blood vessel diseases [111]. Probucol have strong antioxidant properties being able to block oxidative modification of low density lipoproteins (LDL) in vivo [112]. Like other hydrophobic direct antioxidants, probucol suppresses the propagation of ROS, especially free radicals [113]. In addition, it appears that this drug can promote endogenous antioxidants production [114]. Probucol tends to accumulate in fat, therefore it should be expected to specifically deliver its antioxidant effect in fatty liver diseases [115]. Animal studies with probucol, show that this drug ameliorates oxidative stress-related parameters in models of lipid derivate hepatic injury [116]. In clinical trials probucol has been studied alone or associated with other drugs. These trials have been carried out mostly in Iran by Merat et al. [101, 115, 117] who found that the treatment with probucol would be effective against NASH. In the first Merat´s pilot study,

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biopsy-proven NASH patients were treated during 6 months with 500mg of probucol daily. Aminotransferase levels improved significantly [115]. The second Merat´s study was a randomized double-blind controlled study with biopsy-proven NASH patients who were treated with 500 mg of probucol daily or placebo during 6 month. These results confirmed the former [117]. Nevertheless, in both studies no final biopsies were performed. The most recent Merat´s study included biopsies performed before treatment and after twelve month of 500 mg probucol daily, who showed improvements in liver enzymes and necroinflammation biopsy score but not in fibrosis parameters [101]. Finally, Tokushige et al. [118] evaluated the effect of probucol in combination with pantethine, the dimeric and more biologically active form of pantothenic acid (vitamin B5) that among other effects promotes fatty acid oxidation and reduces serum triglyceride. In this trial, biopsy-proven NASH patients with hyperlipidemia were treated with probucol (500 mg/day) and pantethine (600 mg/day) for six months, showing important improvements in aminotransferases. In eight patients, liver biopsy was performed before and after the intervention. Four of these patients showed inflammation improvement, and two showed fibrosis improvement. 3.2.3. N-Acetylcysteine N-acetylcysteine is a thiol compound derived from the metabolism of amino acid Lcysteine. It is an endogenous precursor of glutathione formation with marked direct and indirect antioxidant properties. Its indirect antioxidant activity is related to its ability to stimulate glutathione generation. The direct antioxidant effect is derived from the thiol reductive ability. Several investigations of N-acetylcysteine in NASH animal models show improvements in different pathology markers. Thus, in diet induced NASH rats Thong-Ngam et at. [119] found improvement in levels of total glutathione and decrease in necroinflammation after treating the animals with N-acetylcysteine. In the same animal model Baumgardner et al. [120] demonstrated that N-acetylcysteine improves several markers of NASH progression, including TBARS and activation of cytochrome P450 CYP2E1 isoform, by inhibiting the development of oxidative stress and TNF-α secretion, without blocking the development of steatosis. Only three small clinical trials of N-acetylcysteine in NASH are available. The first study was a controlled trial conducted by Gulbahar et al [121] in NASH patients treated with diet management followed by 600 mg/day of N-acetylcysteine. In this trial, significant improvement in aminotransferase levels was found. Nonetheless, no hepatic biopsies were performed. Later in a randomized placebo study with biopsy-proven NASH patients, Pamuk and Sonsuz [122] found significant decrements in aminotransferase levels in the intervention group after a month treatment period with oral administration of 600 mg/day of Nacetylcysteine. Unfortunately, this study lacks of final biopsies too. Other study, based in combined therapy with N-acetylcysteine (1.2 g/day) and the hypoglycemiant agent metformin (850–1000 mg/day) during a year, was performed by De Oliveira et al. [123] in obese biopsy-proven NASH patients. This trial excluded individuals with comorbidities other than obesity such as hyperlipidemias and diabetes. Biopsies were performed in all patients at the end of the therapy. After the intervention, most biochemical

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and histological NASH parameters showed significant improvement, reducing several markers of NASH, including fibrosis. 3.2.4. Vitamin C Vitamin C or L-ascorbate is a glucose derivate lactone, which humans cannot synthesize. This compound is necessary for a range of essential metabolic reactions (e.g. collagen hydroxylation) [124]. In addition, L-ascorbate is a strong water-soluble reducing agent with important antioxidant functions demonstrated in in vitro [125]. This vitamin, in addition to its direct free radical scavenging abilities, has the property to regenerate oxidized vitamin E and glutathione to the reduced active forms [126]. Some animal studies have shown that vitamin C is able to improve liver antioxidant defenses [127] and others showed that this vitamin can prevent liver induced oxidative damage [128]. Thus, it is reasonable to think that vitamin C therapy could improve NASH. The sole clinical trial testing the effect of vitamin C in NASH patients was the previously mentioned Harrison et al. study [104]. However, in this study it is difficult to establish the contribution of vitamin C alone, as it was assayed in combination with vitamin E. 3.2.5. S-Adenosylmethionine S-adenosylmethionine is an amino acid derived from folate cycle by the reaction of methionine and adenosine triphosphate. It is fundamental in a several group of reactions by its ability to donate a methyl group, these reactions include DNA methylation and phosphatidylcholine synthesis [129]. The indirect antioxidant ability of Sadenosylmethionine would be due to the production GSH through the formation of cysteine. Animal studies of different models of hepatitis show a significant increased of cytosolic and mitochondrial GSH after the incorporation of S-adenosylmethionine [55, 130, 131]. Clinical studies have demonstrated that patients with different liver diseases have a deficient conversion of methionine to S-adenosylmethionine, with the subsequent and decreased levels of plasma and hepatic GSH [132-134]. S-adenosylmethionine has been shown to reverse the decreased GSH hepatic levels in liver disease patients after 36 weeks of oral therapy [135]. In addition, other clinical human studies have shown the benefits of Sadenosylmethionine therapy in different forms of liver disease, including alcoholic steatohepatitis and intrahepatic cholestasis of pregnancy [136-138]. To our knowledge, no published clinical trials to date have used S-adenosylmethionine in NASH. 3.2.6. Betaine Betaine or trimethylglycine is a physiological metabolite of choline that serves as an alternative methyl donor in the homocysteine derivate synthesis of methionine and in the phosphatidylcholine synthesis [139]. The hypothetical indirect antioxidant effects of betaine would be associated with its ability to increase S-adenosylmethionine [71]. Animal models of alcohol-induced fatty liver demonstrate that betaine oral therapy increases hepatic S-adenosylmethionine levels, and prevents the progression of steatosis [140]. Betaine also decreases mitochondrial derivate apoptosis in rat hepatocytes treated with bile acids [141].

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There are very few clinical trials of betaine in NASH. Miglio et al. [142] randomized NAFLD patients to compare the effects of an oral form of betaine glucuronate in combination with nicotinamide ascorbate and diethanolamine glucuronate versus placebo. The intervention group shows improvement in ultrasound scoring of liver steatosis and aminotransferases, compared to the placebo group. However, it was unknown how many patients had NAFLD versus NASH because no biopsies were performed. Other study, an uncontrolled small pilot trial of betaine anhydrous (20 gr daily) in biopsy-proven NASH patients conducted by Abdelmalek et al. [143] for 1 year, demonstrated some liver improvements. Overall fibrosis, necroinflammatory grade and steatosis showed improvements after the final biopsy. Nevertheless, at the end of the trial, serum triglycerides showed a nonsignificantly increase, supporting a possible betaine lipotropic effect. 3.2.7. Genistein Genistein is a phytoestrogen present in soybeans, which has a variety of pharmacological features including, antineoplasic, antioxidant and anti-inflammatory actions. Phytoestrogens are isoflavones, compounds with well known direct antioxidant properties and proven abilities to improve various diseases associated with oxidative stress [144]. Finally, a hypothesis of the role of the as antioxidants therapeutic tools in NAFLD or NASH patients is shown in figure 9-2.

Figure 9-2. Diagram representing the different possible strategies to treat NASH in relation to inhibit direct and indirect mechanisms associated to oxidative injury focusing in antioxidants agents. Dual arrows imply bidirectional relation, T imply direct inhibition and dotted T imply indirect inhibition.

Animal models show improvement of NASH after genistein treatment, and recent in vitro studies revealed that genistein affects proliferation of HSCs and consequently displays antifibrotic effects. For example, Yalniz et al. [145] showed in a recent study that genistein remarkably prevented the emergence of NASH by improving the biochemical and histopathological abnormalities via attenuating oxidative stress. Liver lipid peroxidation levels were significantly higher in the untreated group in comparison to the genistein group, measured by MDA detection. In addition, treatment with genistein improved inflammation parameters decreasing significantly TNF-α plasma levels, a known mediator associated to oxidative stress. Despite the need to conduct clinical trials to demonstrate the therapeutic

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effects of genistein in human NASH patients, it is known that the consumption of soy products rich in phytoestrogens has proved to have numerous favorable effects on various diseases. Moreover, the underlying mechanism of action, based mainly in antioxidant properties, and their animal studies, suggest that this compound, associated to traditional therapy, could be promising in the treatment of NASH patients.

4. Conclusion and Perspectives In conclusion, it is clear that the overabundance of oxidant species is a key step in both, the development of liver steatosis and the progression from simple steatosis to NASH. Hepatic steatosis should not be considered benign because this condition itself plays a major role in NASH development. Hence, NAFLD patients should be monitored for possible prevention of NASH, particularly individuals with comorbid conditions strongly linked to inflammation and oxidative stress, such as obesity, insulin resistance and hyperlipidemia. The missing link between simple hepatic steatosis and NASH is associated with metabolic impairment of the liver due to accumulation of FFAs agents that are strongly associated to oxidant species production by different pathways. The response to these challenges is partly determined by genetic factors associated with the activity of antioxidant defense systems. When these defenses become overwhelmed, oxidative stress is triggered. Consequently, this metabolic derangement causes chronic inflammation, cell death and fibrosis of the liver. Thus increased oxidative stress has been well documented in humans as an important mechanism of progression of statosis consecutively to inflammation and fibrosis. Therefore, a great interest of antioxidant therapies to diminish liver oxidative damage has arisen, since metabolic therapy has not given promising results in improving biochemical markers and liver morphology of these patients. Among the studied antioxidant substances, Vitamin E seems to have one of the best therapeutic effects, likely to be improved in combination with other antioxidants agents, suggesting that the combination of substances with different antioxidant mechanism could be more effective than the use of only one substance. Other potentially therapeutic agents, such as probucol, vitamin C and betaine among others, have been used on the basis of there direct or indirect antioxidant properties. Moreover it should be noted that more clinical trials are currently being performed in order to improve the clinical outcome of these patients.

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[56] Caro AA, Cederbaum AI. Oxidative stress, toxicology, and pharmacology of cyp2e1*. Annu. Rev. Pharmacol. Toxicol. 2004;44:27-42. [57] Zhang, DX.; Zou, AP.; Li, PL. Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries. Am. J. Physiol. Heart Circ. Physiol. 2003;284:H605–H612. [58] Won, JS.; Im, YB.; Khan, M.; Singh, AK.; Singh, I. The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide-mediated expression of inducible nitric oxide synthase. J. Neurochem. 2004;88: 583–593. [59] Macmillan-Crow, L. A.; Cruthirds, D. L. Invited review: manganese superoxide dismutase in disease. Free Radic. Res. 2001;34:325–336. [60] Summers SA. Ceramides in insulin resistance and lipotoxicity. Prog. Lipid. Res. 2006;45:42-72. [61] Boland MP, O’Neil LA: Cermide activates NF_B by inducing the processing of p105. J. Biol. Chem. 1998;273:15494–15495. [62] Lin KT, Xue JY, Nomen M, Spur B, Wong PY. Peroxynitrite-induced apoptosis in HL60 cells. J. Biol. Chem. 1995 ;270:16487-16490. [63] Unger RH, Zhou YT. Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover. Diabetes. 2001;50 Suppl 1:S118-s121. [64] Srivastava S, Chan C. Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line. Biotechnol. Bioeng. 2008;99:399-410. [65] Smith EL, Schuchman EH. The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases. FASEB J. 2008;22:3419-3431. [66] Russell JW, Golovoy D, Vincent AM, Mahendru P, Olzmann JA, Mentzer A, Feldman EL. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J. 2002;16:1738-1748. [67] Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309-1312. [68] Juránek I, Bezek S. Controversy of free radical hypothesis: reactive oxygen species-cause or consequence of tissue injury? Gen. Physiol. Biophys. 2005;24:263-278. [69] Machado MV, Ravasco P, Jesus L, Marques-Vidal P, Oliveira CR, Proença T, Baldeiras I, Camilo ME, Cortez-Pinto H. Blood oxidative stress markers in nonalcoholic steatohepatitis and how it correlates with diet. Scand. J. Gastroenterol. 2008;43:95-102. [70] Wei Y, Clark SE, Morris EM, Thyfault JP, Uptergrove GM, Whaley-Connell AT, Ferrario CM, Sowers JR, Ibdah JA. Angiotensin II-induced non-alcoholic fatty liver disease is mediated by oxidative stress in transgenic TG(mRen2)27(Ren2) rats. J. Hepatol. 2008;49:417-428. [71] Chang CY, Argo CK, Al-Osaimi AM, Caldwell SH. Therapy of NAFLD: antioxidants and cytoprotective agents. J. Clin. Gastroenterol. 2006;40:S51-S60. [72] Videla LA, Rodrigo R, Orellana M, et al. Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients. Clin. Sci. (London). 2004;106:261268.

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In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter X

Neurodegenerative Disorders Rodrigo Pizarro Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile Supported by FONDECYT, grant 1070948

Abstract Oxidative stress has been related to the pathogenesis of virtually every neurodegenerative disease. However, two clinical entities stand out for the major epidemiological burden they provide, as they are intimately related to our increasingly aging population. Alzheimer’s disease and Parkinson’s disease are the two most prevalent neurodegenerative diseases affecting roughly over 5.5 million people in the United States alone. In spite of this, the understanding of their underlying pathophysiological mechanisms is scarce, and thus, they have remained difficult to treat, prevent or cure. Early diagnosis is fundamental, as is in early stages of the neurodegenerative process when therapeutic interventions in both animal models and clinical trials have proven more beneficial. However, early detection can be a painstaking procedure due to their subtle, highly unspecific first clinical features and still rather undeveloped biomarkers. It is in all of these tasks where the understanding of the roles of oxidative stress in the pathogenesis of such conditions has been proved beneficial. Biochemical markers of oxidative stress now seem a feasible way of early detection of such diseases. We could also give possible explanations for the mixed results obtained in clinical trials using antioxidant supplementation against neurodegenerative diseases. The task now is to continue looking deeper at oxidative stress in the mechanisms of such diseases, but also to develop new therapeutic resources to meet the needs of a rapidly growing population. This chapter deals with the general pathophysiology of oxidative stress and its role in the pathogenesis and antioxidant supplementation in the detection, understanding and treatment of Alzheimer’s disease and Parkinson’s disease.

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1. Introduction Neurodegenerative diseases (ND) are becoming ever more prevalent with the increasing amounts of aging population. This new epidemiologic scenario proposes a new endeavor to both clinicians and the scientific community, as the knowledge of the pathogenesis of these diseases is scarce, and thus, have remained challenging to treat, prevent, or cure. Neurodegenerative processes have been revealed in an enormous and heterogeneous set of inherited and sporadic conditions, characterized by two main events: 1. A progressive neuronal loss in the affected central or peripheral nervous system structures, leading to clinically relevant impairment of the cerebral function and; 2. Clinical manifestations of a progressive decline in cognitive (dementia) and/or motor function that is beyond what might be expected from normal aging [1]. 1.1. Normal Physiologic Cognitive Decline The normal aging process is generally associated with a gradual loss of neurons, a lower ability of the brain to create new synapses and various biochemical changes at the membrane level. The latter influences axonal signal transduction, regulation of membrane bound enzymes, ion channel structure as well as the maintenance of various receptors [2]. Also, cerebral blood flow decreases with aging and is associated with a loss of endothelial function [3]. Endothelial cells form a critical component of the blood-brain barrier (BBB) and are actively involved in the transport of nutrients from the blood to the brain [4]. Cognitive deterioration is so frequently observed in the elderly that it is commonly thought of as an inevitable end-point of normal cerebral decline. However, dementia increases its prevalence in an exponential manner up to age 80, where its prevalence begins to drop to completely flatten by age 95, where it stabilizes at 40%. Therefore, neurodegeneration should be considered an age-related- pathological process, distinct from those implicated in normal ageing [5]. 1.2. The Burden of Neurodegenerative Diseases Dementia currently affects over 24 million people worldwide, with 4.6 new reported cases each year. With the increasing aging of the population, it’s to be expected that this figure will double in the next 20 years, up to an estimate of 42.3m in 2020 and 81.1m by 2040 [6]. Older age has been consistently associated with an increased risk of developing dementia [7]. The prevalence of dementia in people age 60 to 64 is roughly 1%, while it can reach well over 30% in people over 85 years old [6]. Dementia represents a leading cause of death in the elderly. It’s a common cause of morbidity and mortality, with 50% of females and 40% of males aged over 84 years dying of dementia [8]. As in any age-related pathology, the shorter life expectancy of men biases lifetime history of the disease and the proportion of death rates between sexes [7]. Dementia has been systematically underestimated in mortality data, as recent studies reveal. Death certificates tend to list the immediate cause of death of patients with dementia as pneumonia, sepsis or trauma while omitting dementia completely. Physicians tend to

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overstate other causes of death, such as cardiovascular events and/or hesitate to determine dementia out of fear of the social pressure, diagnostic limitations or social security issues [8]. Dementia’s associated morbidity and mortality rates can be explained due to its effects on common physiologic processes such as swallowing. Successful swallowing requires the input from cortex, sub cortex, brain cortex and cranial nerves [10]. The alterations in the process of swallowing can thereby predispose the person with dementia to dehydration and/or malnutrition, and even acquire and die from aspirative pneumonia [9, 10].

Figure 10-1. Sources of reactive oxygen species involved in the pathogenesis of Alzheimer’s disease. There is sufficient evidence to support the establishment of oxidative stress before the deposition of the Aβ plaques (primary insult?). Aβ-peptides are generated from APP. Aβ peptides as well as APP have been found inside mitochondria and included in membranes where they increase ROS production by complex I uncoupling. However, Aβ peptides are also ROS generating agents per se. Other sources of ROS are metallic ions (Fenton reaction), and NADPH oxidase. Oxidative stress leads to grave macromolecular damage and microglia activation, which leads to further oxidative stress and proimmflamatory chemokine production. Reactive oxygen species and inflammation promote NF-κB activating pro-inflammatory genes perpetuating the oxidative stress-inflammation cycle until the cell sustains irreversible damage to progress into neurodegeneration.

1.3. Diagnostic Procedures and Potential Treatments Even though the sensitivity of clinical diagnosis can reach 93% in skilled hands, its reported specificity is much lower, at 55% [11]. This has left dementia as a severely under diagnosed entity. The recognition of dementia at early stages or its less frequent presentations can be even more challenging on solely clinical basis. With the introduction of potentially successful treatment strategies, the need for a reliable biomarker has become even more pressing. New neurochemical determinations are now being tested to detect the disease at its

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initial stages, where interventions have been more promising. These procedures should also aid in the process of making a correct differential diagnosis of the disease in its unusual presentations [12]. Several relevant alterations have been reported regarding the poorly understood pathogenesis of neurodegenerative disorders. Among them, the role of oxidative stress has been well established as one of the primary events in the neurodegenerative process [13-15]. Moreover, the evidence regarding the attack by reactive species of oxygen (ROS) and nitrogen (RNS) on lipids, proteins and DNA has been found in virtually every type of brain disease, as well as in the physiologic aging process [1]. 1.4. The Role of Oxidative Stress in Neurodegenerative Diseases Reactive oxygen species are constantly being produced by normal cell metabolism [16]. In normal cell functioning, there is a balance among the ROS production and the antioxidant defense. If this thin balance is altered, due either to excess in the ROS production or a deficit in the antioxidant defense, oxidative stress is established. Depending on the magnitude of this phenomenon, there are several possible cellular responses. Moderate oxidative stress leads to the activation of cell proliferation and protein induction mechanisms with contained macromolecular damage. In massive ROS production and liberation there is inevitable damage of macromolecules such as lipids, proteins, RNA, and DNA, which has been shown to be involved in the etiology, and/or progression of multiple human diseases [17-19]. Macromolecular damage leads to cellular malfunctioning and if the sustained injury reaches a point of no return, the cell enters programmed death or necrosis [20, 21]. It has been well established that the mitochondria is a major contributor of ROS production and thus, tissues with high metabolic rate are in need of high antioxidant protection, to cope with these exigent requirements [21]. There would only be a ROS handling deficit under massive pathologic hyper-production as occurs in mitochondrial dysfunction as well as hypo-metabolism and/or when ROS are supplied from external sources as occurs in pathologic conditions. Therefore, these entities have been related to the pathogenesis of neurodegenerative procecess such as AD [17]. In hypoxemia, for example, there is a raise on the mitochondrial ROS production, by the effect of Ca+2 overload and other mechanisms, leading to an augment in superoxide (O2•–) that surpasses the antioxidant defense [20-22](for further general aspects of oxidative stress, refer to Chapter 1). Cerebral tissue appears particularly susceptible to oxidative stress-mediated injury. The central nervous system (CNS) has a high metabolic oxidative rate. While accounting for only 2% of the body weight, 20% of the resting body oxygen consumption is destined to cerebral functioning [1]. At the same time, the brain is composed mostly by post-mitotic neurons, and has a very high content of substances susceptible to oxidation such as polyunsaturated fatty acids (PUFA) and catecholamines. It also posses a high content of transition metals and ascorbate levels, which act together as potent pro-oxidants, coupled to relatively limited antioxidant capabilities. Thus, the brain is quite exposed to the attack of oxidative stress [1, 19, 23]. For many years researchers have been trying to elucidate the precise role of ROS on the pathogenesis of neurodegenerative disorders. However, as the pathogenesis of such diseases

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has remained elusive, treatment and prevention strategies have not been successful in modifying their natural history, or considerably diminishing their symptoms in order to improve the quality of life of their victims [19]. There are several hypotheses to explain the mechanisms of ND. Among these, the role of oxidative stress has been gaining strength, as it encompasses the action of many other agents such as redox active trace metallic elements, mitochondrial dysfunction and oxidative injury through pro-oxidant peptide (β-amyloid) mechanisms [24]. Over the last decade, free-radical-mediated damage has been associated with virtually all stages of AD [19, 25]. Data from clinical trials and experimental models suggests that the pathologic expression of AD’s characteristic proteins is related to the establishment of oxidative stress per se leading to increased membrane lipid peroxidation [26-28]. This chapter will focus on the role of oxidative stress in the pathogenesis of ND and available evidence to sustain the use of redox-state modifying strategies. In order to achieve a clearer understanding of the matter at hand, Alzheimer’s and Parkinson’s disease will be analyzed separately from further on.

Alzheimer’s Disease Alzheimer's disease (AD) is the most frequent neurodegenerative disorder associated with the onset of dementia in the elderly. It’s the most common form of dementia in adults over age 65, and the fourth leading cause of death in the United States, currently affecting over 4.5 million Americans. The mean expected lifespan following diagnose is up to 8.5 years with a range of 1–25 years [29-31]. Alzheimer’s disease is a mainly sporadic disease, where ageing represents the main risk factor [32]. Studies reveal that up to 5% of AD could be caused by missense mutations for either Alzheimer β-amyloid (Aβ), precursor protein (APP) or some of the enzymes involved in its metabolism [12]. Alzheimer’s disease presents a cognitive and memory impairment mostly derived from hippocampal malfunctioning, leading progressively to the dramatic and socially disabilitating condition of the well known, late stages of the disease. Clinical features of AD include anterograde amnesia and loss of language, motor skills, abstract reasoning, concentration and executive function that have a substantial effect on daily functioning (table 10-1) [33]. Mild cognitive impairment (MCI), as first described by Petersen et al [34] (table 10-2), is defined as a cognitive decline of the individual which is more pronounced that what should be expected for his/her age and education level, but does not interfere in a marked manner with the activities of daily life [35, 36]. There is a well-established augmented risk for patients with MCI, who are up to 10 times more likely to develop AD than the general population. Progression from MCI to early AD dementia increases annually at a rate of 10-12% per year in contrast to the 1-2% of general population. Conversion to dementia is finally 80% by the sixth year of follow-up, with only 5% of patients remaining stable or reverting to normal after the sixth year of follow up [3739]. Early AD dementia can be diagnosed according to the criteria described on table 2. Further progression of the disease leads to late-stage AD (LAD) characterized by severe dementia with profound global cognitive deficits and motor compromise [19].

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Table 10-1. National Institute of Neurological and Communicative Diseases and Stroke/Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) Diagnostic Criteria for Alzheimer’s Disease [35]. Symptoms Dementia diagnose*1 including clinical dementia rating scale score of 0.5-1 Decline in one or more areas of cognition*2 Progressive worsening of cognitive function*3

Exclusion criteria Disturbance in consciousness Onset age outside 40-90 Systemic or neurological condition that could otherwise explain dementia

Impaired ADLs

*1 Established by clinical evaluation and mental status tests. *2 In addition to memory. *3 From a previous higher level.

Table 10-2. Diagnostic Criteria Required for the diagnose of Mild Cognitive Impairment [34]. Symptoms Memory complaints Objective memory impairment*

Preserved functions Intact general cognitive function Intact activities of daily living (ADLs) Clinical exclusion of dementia

*According to age and education based on a score of 1.5 standard deviations from the mean of controls on the CERAD Word List Learning Task [182].

2. Pathophysiology 2.1. Histopathology and Molecular Biology Hyperphosphorylated forms of Tau proteins and Aβ peptides are responsible for the classic histological hallmarks of the disease: the presence of neurofibrillary tangles (NFT) and the accumulation of cerebral senile plaques (SP) of Aβ deposit. These findings are also accompanied by loss of neurons (cholinergic in particular) and synapses, in the forebrain, and depletion of neurotransmitter systems in the hippocampus and cerebral cortex. Other frequent histological findings are neuropil thread formation, proliferation of reactive astrocytes in the entorhinal cortex, hippocampus, amygdala and association areas of frontal temporal, parietal and occipital cortex [12, 19, 33, 40]. The etiology of the disease is still unknown, but current hypotheses focus on synaptic and neuronal loss and the role of amyloidogenic and/or Tau proteins tightly collaborating with oxidative stress, mitochondrial and vascular dysfunction as primary events of the disease [41, 42]. Tau is a neuronal protein present in axons and dendrites where it promotes tubulin polymerization and stabilizes microtubules and thus contributes to cell structure, axon transport and cellular growth. Neurofibrillary tangles consist of intracellular filaments deposits of the hyperphosphorylated form of Tau protein. The hyperphosphorilation of Tau

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prevents the protein from binding to microtubules, causing destabilization of cell structure and thereby, likely contributing to the loss of axons, dendrites and synapses [43-45]. Alzheimer β-amyloid is a 39–42 amino acid peptide generated by sequential proteolytic cleavage of amyloid β-protein precursor protein (APP), a large transmembrane glycoprotein that is initially cleaved by the β-site APP cleaving enzyme 1 (BACE1) and subsequently by membrane bound proteolytic enzymes, called secretases in the transmembrane domain [4648]. β- and γ-secretase, are the two main proteases that cleave APP at the amino and carboxyl-terminus of the Aβ peptide, respectively and are hence, directly responsible for Aβ peptide generation. On the other hand, a less abundant α-secretase promotes a nonamyloidogenic pathway of APP proteolysis. Alpha-Secretase activation may even have the additional advantage of generating the putatively neuroprotective sAPP-α, as well as preventing neurotoxic Aβ peptide formation [49]. Its potential therapeutic properties will be discussed further on. The resulting length of the Aβ protein is dependent on initial cleavage of the extracellular domain generating the amyloidogenic end products Aβ 1-42 and Aβ 1-40 [50]. Low molecular weight oligomers, particularly Aβ 1-42 have been found to be the most toxic for neurons and form aggregates that appear to be the predominant species in SP. The ratio between Aβ 1-42 and Aβ 1-40 in CSF correlates directly with the onset age of AD [51]. It has been suggested that Aβ peptides might have a toxic effect, and impair synaptic plasticity long before its SP formation and deposition [52]. Aβ appears to promote neuronal death, at least in part by the generation of oxidative stress. Indeed, this process seems to be dependent of the predominant β-sheet conformation. It has been proposed that soluble Aβ might be the initial event that triggers the neurodegenerative cascade. However, whether oxidative stress is cause or consequence of the amyloid deposition is still in dispute [53]. It is also highly unlikely to be the sole element contributing to the progressive nature of the disease. Nevertheless, the exact mechanism through which Aβ can interfere with normal synaptic physiology and contribute to cognitive deficit through a preferentially basal forebrain cholinergic cell loss is yet to be determined [44]. Senile plaques can be differentiated into two histological forms; a) diffuse plaques of amorphous extracellular neurite lacking, deposits of Aβ or b) neuritic plaques, composed of extracellular deposits of insoluble Aβ surrounded by dystrophic neurites, activated astrocytes and microglia. Recent studies suggest that Aβ can impair synaptic plasticity through mechanisms that might contribute to cognitive decline in AD. Evidence is mounting that Aβ oligomers can mediate these effects, possibly accounting for why plaque number is such a poor predictor of cognitive impairment. The deposit of SP is a frequent finding in the elder, not always linked to cognitive decline. Early studies suggested that there was a direct relation between the deposit of SP and cognitive decline. Further studies have shown that the presences of NFT and synapse loss are better histological markers of the disease. Indeed, the density of terminals containing the acetylcholine-synthesizing enzyme, choline acetyltransferase in the neocortex shows a negative correlation with the severity of dementia and is already altered in the early disease [17]. Interestingly, there is a direct link between the function of cholinergic neurons, the cerebrovascular system and amyloid pathology. Acetylcholine is a potent vasodilator affecting cerebral blood flow [54]. Amyloid deposits are also found around cortical vessels

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where they promote Aβ deposition that consequently contributes to brain hypoperfusion or vice versa [40]. Also, endothelial dysfunction of cells participating in the BBB may contribute to inadequate supply of nutrients to the brain. Indeed, increased hypertension, leading to augmented BBB permeability in cortex and hippocampus has been directly associated with Aβ deposit in murine models [55]. Current evidence also supports a role for cholinergic innervation in the non-amyloidogenic maturation of APP, whereas amyloidogenic related peptides depress the activity of cholinergic neurons. These cholinergic neurons innervate the hippocampus and neocortex, being central to the loss of cognitive function in AD. It has been shown that Aβ peptides may have acute detrimental effects on acetylcholine synthesis and release and are neurotoxic on the long term [40]. For a long time researchers have been trying to elucidate If Aβ and Tau modifications are related, or represent parallel mechanisms in the pathogenesis of AD. There’s evidence to support that Aβ toxicity can induce Tau phosphorylation and production of 17 kD Tau fragments. Moreover, there’s evidence that Tau-deficient neurons might be more resilient to Aβ toxicity independently of Aβ deposit, or dystrophic neurites [56]. Thus, Tau appears to be essential for the Aβ-mediated neurotoxicity, linking the two pathological features mechanistically to the loss of cholinergic neurons in AD [57]. Also very interesting are recent findings revealing that NFT and Aβ may be linked by their interaction through the Wnt pathway [58]. 2.2. Oxidative Stress in Alzheimer’s Disease Multiple studies have underlined the importance of oxidative stress in the pathogenesis of several ND diseases such as AD. However, the origin of the initial insult has remained elusive to these days. Numerous studies have shown evidence of increased levels of oxidative damage in brain tissue and cerebrospinal fluid (CSF) of patients with ND [59]. We will explore the source of ROS in the CNS and the antioxidant endogenous defense, to finally review the most relevant biological markers of oxidative stress. 2.2.1. Sources of Reactive Oxygen Species Besides ROS produced in the CNS from normal and altered mitochondrial function, and the presence of transition metals, also recent studies now recognize the enzyme NADPH and Aβ peptides per se as very relevant ROS producing agents [60, 61]. It is possible that all these elements interact, especially at early stages of the disease, where Aβ could enter the mitochondria increasing ROS generation. Recent post-mortem AD studies and transgenicmice studies have revealed that APP can be found in mitochondrial membranes where they can disrupt the electron transport chain leading to oxidative stress-mediated irreversible cell injury [60]. There is evidence to support the role of APP and Aβ in this organelle malfunction as they are both present in the mitochondrial membrane, interacting with mitochondrial proteins and disrupting its import channels, intracellular transport and altering the electron transfer chain, leading to a ROS overproduction and further mitochondrial damage [62]. Recent evidence supports the establishment of oxidative stress before the appearance of Aβ plaques. Researchers have found that levels of 8-hydroxyguanosine (8OHG), an oxidized

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derivative of guanosine, and protein nitrotyrosilation, are quantitatively increased at early stages of the disease, and decline with disease progression. Also there was an inverse relationship between histological findings (Aβ deposition and NFT formation) and duration of dementia. Furthermore, oxidative stress was found to be much lower in familiar forms of AD (ApoE e4 allele) and Down syndrome, in which abundant Aβ deposits were found. All these findings support the role of oxidative stress as a putative primary event in the pathogenesis of the neurodegenerative process [13]. Alzheimer β-amyloid deposits have been related to the activation of microglia through binding to scavenger receptors, which leads to a pro-inflammatory response, characterized by the release of cytokines and ROS. Microglial activation also leads to an increase in the clearance of Aβ deposits, compatible with the role of oxidative stress as a primary event in the pathogenesis of AD [63, 64]. In addition to the Aβ induced oxidative stress and inflammatory damage by microglia activation there’s also free radical production though other sources such as Fenton reaction. All this oxidative stress on the neuron leads to DNA and RNA oxidation, lipid peroxidation of biological membranes to produce 4hydroxynonenal (HNE), malondialdehyde (MDA) and protein oxidation [17, 65, 66]. Prooxidative state also promotes NF-κB activation leading to the transcription of proinflammatory genes. These are responsible for the increased pro-inflammatory cytokine release and activation of endothelial cell adhesion molecules such as 1,25 monocyte chemoattractant protein-1,26 and others related to atherogenesis [67]. 2.2.2. Antioxidant Defense Immunohistochemical studies have revealed local increases in CU/Zn superoxide dismutase (SOD) and catalase over NFT and SP against age-matched controls [68]. The antioxidant enzyme hemeoxygenase-1 has been shown a local increase at NFT [69]. Also as a response to oxidative damage, DNA repair in AD has shown increased levels of excision repair cross-complementing gene products, apparently in an attempt to increase DNA repairing capacities [70]. However, during the progression of the disease, DNA repairing capacity might become overwhelmed [71]. Glutathione (GSH) is the most abundant peptide in the CNS. This tripeptide is present predominantly in its reduced (GSH) form, playing a fundamental role in defense of brain cells against oxidative stress as a potent intra and extracellular antioxidant, located in epithelial glial cells and neurons. Apart from its numerous essential cellular functions it also plays vital roles in the nervous system, such as regulation of glutamatergic transmission and protection against glutamate-induced exitotoxicity, and participates in the intracellular Ca+2 homeostasis through the activation of calcium-sensing receptors. It also protects the neuron against Ca+2 mediated cell death, antagonizes neuronal apoptosis, redox modulation of several ionotropic receptor currents. It also acts possibly as an excitatory neurotransmitter at sites of its own [72]. The existence and location of these putative GSH receptors has been described in brain cortex and spinal cord, however not yet in the hippocampus [73]. With such critical roles in the CNS, its role in the pathogenesis of various ND such as AD, schizophrenia and Parkinson disease seems feasible [74].

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Glutathione participates in the modulation of taurine release through complex regulation of several ionotropic glutamate agonists. Taurine acts as an inhibitory neuromodulator and shares common features with GSH. Taurine is also critical during SNC development, participating in functional maturation and cell migration in the cerebellum. There’s evidence that shows that GSH’s neuroprotective and plastic functions are only possible through its interaction with taurine. Both GSH and taurine share neuroprotective roles and interact critically in the developing rodent hippocampus. At low extracellular concentrations (μM), GSH is capable of inhibiting the NMDA receptor-mediated taurine release, and therefore affect the development of excitatory neural circuits in the hippocampus. However, at higher concentrations (mM), GSH enhances kainate-evoked taurine release, providing an exitotoxicity-attenuating effect. Therefore extracellular GSH, in collaboration with taurine, appear as essential neuroprotectors during neuronal development. Glutathione is an important antioxidant, and taurine an inhibitory neuromodulator playing a collaborative role at such critical stages [75]. It has also been reported that in the striatum of adult mice, reduced (GSH) and oxidized glutathione (GSSG) may also have an exitotoxicity-attenuating effect by preventing excessive dopamine depolarization-evoked release and the toxic effects of high glutamate, and by modulating the redox state of Ca+2 channels [72]. Glutathione additionally seems to upregulate the glutamatergic-evoked release by regulating the redox state of both non-NMDA and NMDA receptors. It is thought that the regulation of striatal dopamine release by GSH may have a neuroprotective role, while preserving normal neurotransmitter release, relevant to extrapyramidal motor functions [73]. As a consequence of high magnitude, sustained oxidative stress, the antioxidant defense may become surpassed: GSH gets depleted and/or GSSG augments and therefore, the GSH/GSSG ratio tends to decrease leaving the cell exposed to oxidative stress induced irreversible damage. The GSH/GSSG ratio is a sensitive marker of intracellular oxidative stress [16, 76]. 2.3. Animal Models of Alzheimer’s Disease Since the early nineties, several animal models have been successful in reproducing some of the pathologic hallmarks of AD. In fact, most of the data from clinical studies has been validated by experimental data on transgenic mice models [23]. The transgenic (Tg) 2576 mouse model of AD-like amyloidosis manifests evidence of increased brain lipid peroxidation, before the surge in Aβ levels and deposition [77]. In the same model, there is a concomitant increase in nitrogen reactive species and antioxidant enzymes with the onset of the Aβ deposit [78]. Similarly, APP and presenilin 1 (PS1) double knock-in mice (APP/PS1) manifest features of lipid and protein oxidation at early stages of their phenotype [79]. Also, deletion of the prostaglandin E2 receptor in the double mutant APP/PS1 mice, results in less brain lipid peroxidation and a significant decrease in Aβ levels and deposit [80]. Further recent evidence shows that in a Tg2576 AD-like Aβ mouse model, the use of a thromboxane (Tx) receptor antagonist, blocks the directly delivered into the brain isoprostane iPF (2α)-III induced Aβ levels and deposit. This suggests that Tx receptor activation mediates the effects

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of iPF (2α)-III on Aβ. This hypothesis was supported by cell culture studies that showed that Tx receptor activation increased Aβ and secreted APP ectodomains [81]. In a recent triple transgenic murine model (3xTg-AD), which develops SP and NFT, levels of antioxidants are decreased and by contrast, lipid peroxidation is increased before the appearance of AD-like pathology [82]. In APP transgenic mice, crossed with Mg-SOD heterozygous deficient mice, increased brain lipidperoxidation, has been associated to a significant raise in Aβ levels and plaque deposition [83]. Also, the cross of alpha-tocopherol transfer protein knockout mice with a AD transgenic model mice (Tg2576) manifests an earlier, more severe cognitive dysfunction, associated to increased Aβ deposits, succesfully ameliorated by vitamin E supplementation [84]. An extensive variety of biomarkers of oxidative stress have been related to the pathogenesis of AD. All these signature markers have been used to support the “oxidative stress hypothesis” of AD. A simplistic approach to this hypothesis would sustain that antioxidant supplementation should suffice to stop the ND progression. Conflicting data regarding this aspect have generated a wave of criticism towards the validity of the hypothesis, in need to be clarified [23]. 2.4. Biological Markers of Alzheimer’s Disease As most promising effects of treatment against AD have been found when the intervention takes place at early stages of the disease, there is a pressing challenge to diagnose it as soon as possible. However, as the disease has a multifactorial pathogenesis it has not been a simple task [16]. With the increasing prevalence of the disease, clinicians require the development of reliable diagnostic testing in AD. The general consensus was that sensitivity and specificity for such instruments shouldn’t be underneath 85% and 75% respectively and of course, that it could be used in vivo [85]. In order to achieve this, a peripheral marker (i.e. blood or CSF) to assess the biochemical alterations of the disease would be also highly desirable. 2.4.1. Protein Oxidation Proteins are abundant in peripheral blood and targets for ROS oxidation. The advantages of using protein oxidation in comparison to other markers can be explained due to their early formation and greater stability. Protein oxidation is an early event in oxidative stress and its degradation process takes hours to days in comparison to the few minutes taken for the removal of damaged lipids [16]. The half-life of carbonyl groups in peripheral blood is larger than markers of lipid peroxidation such as MDA [86]. Increased levels of protein carbonylation have already been reported in other diseases related to increased oxidative stress such as diabetes, inflammatory bowel disease, chronic renal failure, sepsis and arthritis [17, 86]. Increased plasma levels of protein carbonylation can also be observed in MCI, and to a greater extent, in AD in comparison to healthy age-matched controls, product of the oxidative stress-inflammation of the disease. In AD and MCI, protein oxidation mostly occurs in the inferior parietal lobe, the cortex and hippocampus. This is not the case in the cerebellum where Aβ deposit is scare. In other words, protein oxidation occurs in brain

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regions where major Aβ deposit can be found during the advanced disease [87, 88]. Levels of 3-nitrotyrosine, another marker of protein oxidation, are elevated in brains of MCI subjects compared with controls, who also exhibit oxidative modifications of several specific protein enzymes, including a-enolase and glutamine synthase [89, 90] The accumulation of oxidized proteins through the loss of their catalytic properties or by interruption of regulatory pathways reflects a surpassing of the cellular repairing capabilities by oxidative stress [91]. 2.4.2. Lipid Peroxidation Poly-unsaturated fatty acids, compounds particularly rich in double bonds, are very susceptible to lipid peroxidation [66]. Post-mortem human studies have shown increased lipidperoxidation in the brain and CSF of AD victims compared to controls, as revealed by several markers such as MDA and 4-hydroxynonenal [65]. F2-isoprostanes (F2) are prostaglandin compounds, also product of the ROS-induced PUFA oxidation. These compounds have been found elevated in typical lesion sites and CSF of AD and MCI patients, compared to age-matched controls [92]. Furthermore, increased levels of 4hydroxynonenal and F2 have been found in plasma of AD patients and thus, the possibility of using peripheral blood determinations seems feasible [93]. 2.4.3. Glutathione Another already discussed viable marker of systemic redox state that can be determined in peripheral blood is the GSH/GSSG relation. It has been previously validated as a useful risk indicator in several human diseases including cancer, atherosclerosis, diabetes, and preeclampsia [16, 17]. 2.4.4. DNA Damage Even though all macromolecules are susceptible to the attack of ROS, in particular to OH, DNA is thought to be especially sensitive. Of the two types of DNA, nuclear (nDNA) and mitochondrial (mtDNA), the latter is more unprotected, as it is closer to ROS sources, lacks the protection of histones and possesses much more limited reparative capabilities. These macromolecules are exposed to strand breakage, DNA-DNA and DNA-protein crosslinking and DNA base modifications such as those derived from oxidation. The most commonly used marker of DNA damage is 8-OHG, product of C8 hydroxylation of guanosine [19]. There’s also evidence of augmented levels of 8-OHG in both mtDNA and nDNA in the brain of AD victims at initial stages (in MCI) [94]. These findings are also coherent to the hypothesis that oxidative stress might play a primary neurodegenerative role. 2.4.5. Hyperhomocysteinemia Hyperhomocysteinemia (hHcy) has been associated with increased thrombogenicity, oxidative stress, and activation of pro-inflammatory pathways, impaired endothelial function and atherogenesis [95]. Hyperhomocysteinemia increases oxidative stress by the induction of

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NADPH oxidase and iNOS activity, but also by the impairment in the function of SOD and glutathione peroxidase (GSHpx). Additionally, oxidation of homocystein leads to further ROS generation [96, 97]. However, the role of hHcy as an independent cardiovascular risk factor and the efficacy of homocystein lowering against atherosclerosis have been recently questioned by major clinical trials [98-100]. Even though both studies successfully and significantly reduced homocystein levels by similar schemes of supplementation with folic acid, vitamin B6, and vitamin B12, the HOPE trial was unsuccessful in decreasing the risk of dying from cardiovascular disease in a primary prevention setting [99]. Neither the NORVIT trial was capable of lowering the risk of recurrent cardiovascular disease [100]. There have been no reports yet on randomized trials of folate and/or vitamin B supplementation in relation to dementia or AD as outcomes [101]. Even though some epidemiological studies have suggested that hHcy is an independent risk factor for MCI, dementia and AD, results have not been consistent [102]. Some studies have shown higher homocystein plasma levels in AD patients [101, 103]. A prospective study also indicated that hHcy is a strong, independent risk factor for the development of dementia and Alzheimer's disease over an 8-year period in the Framingham Heart Study population [104]. Other studies, however have found no significant association between plasma homocystein levels and AD [105, 106]. Elevated homocystein levels have been associated to cognitive decline through both vascular and non-vascular mechanisms [107], and with hippocampal atrophy and cognitive decline in absence of cerebrovascular disease [108]. In another neuroimaging study, higher plasma Hcy levels were associated with smaller frontal and temporal lobe volumes and the presence of silent brain infarcts at MRI, even in healthy, middle-aged adults [107]. Also, rats exposed to hHcy develop deficits in spatial learning and hippocampal signaling [109]. With the current evidence, the relation between hHcy and AD seems controversial, and thus, it does not appear as reliable as other biomarkers. 2.4.6. Asymmetric Dimethylarginine Hyperhomocysteinemia is frequently associated to increased asymmetric dimethylarginine (ADMA) levels [67]. Asymmetric dimethylarginine is an endogenous competitive inhibitor of NOS, derived from its substrate L-arginine, inhibiting the NO production. Asymmetric dimethylarginine has been determined as an independent risk factor of cardiovascular disease [110]. Significant increase in the ADMA levels has been reported in patients with AD, independent of other elements such as gender, age, BMI, hypertension, hyperlipidemia, renal function, tabaquism and diabetes [111]. Cerebrospinal fluid (CSF) ADMA levels have been reported decreased in AD, related inversely to cognitive impairment [111, 112]. This suggests that the blood-brain barrier remains intact in AD and therefore CSF and ADMA levels are regulated independently [111]. Decreased ADMA levels might lead to increased NOS activity and NO production, leading to increased peroxynitrite formation that might explain the extensive protein oxidative damage reported in AD. Another hypothesis proposes that elevated systemic ADMA levels may collaborate in the pathogenesis of AD through chronic cerebral hypoperfusion. Therefore, ADMA should be considered a reliable predictor of AD [111].

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In sporadic as in late-onset familiar AD, ApoE e4 allele has been characterized as a major genetic risk factor, that lowers the average onset age, decreases neuronal metabolism, and increases Aβ deposit [13]. This apolipoprotein binds directly to Aβ promoting fibrillogenesis [113]. Prodromes such as decreased glucose metabolism have been found in AD victims more than two decades before the onset of the disease. Also metal binding properties have been related to isoforms of ApoE [13]. Mutations in the gene encoding amyloid APP and in genes of presenilin-1 and -2 (PS2) have been found to play crucial roles in the pathogenesis of early-onset familial AD [101]. Mutations in the APP gene result in early onset autosomal dominant AD. Mutations in presinilisn also cause early onset autosomal dominant AD [113]. Presinilin-1 is an integral membrane protein that forms the catalytic core of the γ-secretase complex, and is thus related to the accumulation of the Aβ 1-42 protein. The association of an intronic polymorphism (rs165932) of the PS1 gene with late-onset AD has been documented. However, contradicting results have been shown in different populations [114]. It seems that the PS1, PS2 genotype might confer a small level of risk of causing AD in the European population, but results have remained conflicting [115]. 2.4.8. Cardiovascular Risk Factors The roles of cardiovascular risk factors such as atherosclerosis, hypercholesterolemia, hypertension, insulin resistance syndrome, and diabetes mellitus on the pathogenesis of AD are yet to be clarified. There are some elements such as Apo E4 allele, associated with hypercholesterolemia and coronary artery disease, with a well-established strong risk factor for AD. However, there is growing evidence that their mechanisms in AD might differ from those of atherogenesis. On the other hand, there has been mixed results regarding the relation between other cardiovascular risk factors implicated in AD such as hHcy, cholesterol levels and the impact of statins on the development of AD [101].

3. Antioxidants in Alzheimer’s Disease 3.1. Statins Well-established antioxidant properties have been attributed to statins. One of the proposed mechanisms to explain such benefits is the prevention of Rac-translocationmediated NADPH oxidase activation [116]. The use of statins has been associated in some epidemiologic studies with reduced risk of Alzheimer disease (AD). Recently, the use of statins has also been associated with decreased typical AD pathological findings [117]. The Adult Changes in Thought (ACT) revealed a diminished risk for dementia, for subjects who begun statin intake before the age of 80 [118]. However, a large randomized placebocontrolled trial, with onset of the intervention at 65 years old, did not reveal a relationship between statin use and subsequent onset of dementia or AD [119].

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3.2. Antioxidant Supplementation Of the therapeutic approaches to AD, one of the most though of, yet underestimated, is the role of general nutrition. The brain is acutely influenced and maintained by our diet. Approximately 60% of this organ’s dry weight is composed of lipids [1]. The aging cellular membrane is characterized by higher levels of cholesterol, decreased cholesterol turnover and decreased levels of PUFA. This may be related to poor uptake of PUFA over the BBB, decreased incorporation into the membrane and/or reduced enzymatic activity [2]. Fats obtained from our diet directly affect the composition and structure of cell membranes [120]. Obtaining data from the Washington Heights-Inwood Columbia Aging Project (WHICAP), a total of 2,258 community-based non demented individuals were evaluated regarding the following of a mediterranean diet consisting of a higher intake of vitamin C, vitamin E, flavonoids, unsaturated fatty acids, fish, higher levels of vitamin B12 and folate, modest to moderate ethanol intake, and lower total fat consumption. Adherence to mediterranean diet was associated with a significant reduction in the risk for AD [121]. Even though epidemiological studies have reported conflicting data, the evidence to date supports a contribution of nutrition in the risk, and particularly prevention of AD. Increasing evidence reveals that nutrients are far from being a mere energy substrate, rather, stimulating neuronal plasticity, and ameliorating ongoing neurodegenerative processes [40]. 3.2.1. Animal Models Several studies using antioxidants have been also been published in AD transgenic mice models, all of which show a consistent beneficial effect towards their behavioral and amyloidotic phenotype. Experimental thiamin deficiency, an established model of altered oxidative metabolism, exacerbates amyloid pathology in Tg19959 mice, by inducing oxidative stress [122]. Dietary copper, by stabilizing brain SOD activity, reduces Aβ production in APP23 transgenic mice [123]. On the contrary, dietary aluminum modulates brain amyloidosis by increasing oxidative stress in Tg2576 mice [124]. Curcumin, is a well-know antioxidant and anti-inflammatory compound, (a polyphenol) found in Turmeric, the powder obtained from the root of Curcuma longa, frequently used in Indian cuisine. Curcumin treatment, in low-dose (160 ppm) and high dose (5000 ppm), significantly lowered oxidized proteins and interleukin-1beta, in Tg2576 mice brains. With low-dose, but not high-dose curcumin treatment, the astrocytes marker glial fibrillary acidic protein (GFAP) was reduced, and insoluble Aβ, soluble Aβ, and plaque burden were significantly decreased by 43-50%. However, levels of APP in the membrane fraction remained unchanged [125]. Also in Tg2576 mice, vitamin E supplementation, early during the evolution of their disease phenotype (before SP are deposited), showed quite revealing results. One group of Tg2576 mice received vitamin E starting at 5 months of age until they were 13 months old, the second group started at 14 months of age until they were 20 months old. Brain levels of 8,12-iso-iPF2alpha-VI, a specific marker of lipid peroxidation, were significantly reduced in both groups of mice receiving vitamin E compared to placebo. Trangenic mice Tg2576 administered with vitamin E at a younger age showed a significant reduction in Aβ levels and

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amyloid deposition. By contrast, mice receiving the diet supplemented with vitamin E at a later age, when amyloid plaques are already deposited, did not show any significant difference in either marker when compared with placebo. These results support the hypothesis that oxidative stress is an important early event in AD pathogenesis, and antioxidant therapy may be beneficial only if given at this stage of the disease process [126]. Numerous studies have shown that Melatonin is decreased during the aging process and that AD patients suffer a profound reduction of this pineal hormone. It has also been demonstrated that melatonin protects neuronal cells from Aβ-mediated oxidative damage and inhibits the formation of β-sheets and amyloid fibrils. Again in Tg2576 mice, administration of melatonin partially inhibited the expected time-dependent elevation of Aβ, reduced abnormal nitration of proteins, and increased survival [53]. Also in wild type (WT) mice, intraperitoneally injected with Aβ 25–35 peptides, melatonin therapy significantly enhanced the activities of oxidative scavenging enzymes such as SOD, catalase and GSH levels in the astrocytes, lymphocytes and hepatocytes. Immunohistochemistry studies reveal that melatonin prevents the activation of GFAP in neocortex and transcription factor NF-κB in liver and neocortex of Aβ injected mice. It also prevents the elevation of dopamine depletion and its degradation products. These findings suggest that melatonin could act as a protective agent against oxidative damage in AD by scavenging ROS and thus by maintaining the activities of the antioxidant enzymes, regulating the GSH levels, and preventing Aβ-induced dopamine turnover [127]. (−)-Epigallocatechin-3-gallate (EGCG), the main flavonoid in green tea has been found to decrease Aβ-mediated toxicity through an increase in secreted levels of the soluble form of APP (sAPP-α) in Tg APP overexpressing mice. In further experiments, EGCG or placebo was administered to mice of the same type, at 8 months of age for 6 months. Several histopathological analyses revealed that plaque burdens were significantly reduced in the cingulate cortex, hippocampus, and entorhinal cortex. ELISA of brain homogenates revealed consistent reductions in both Aβ 1–40 and 1–42 soluble and insoluble forms. In the cognitive experiments, animals treated with EGCG also showed better performance in comparison to placebo [128]. Retinoic acid (RA) has been shown to control the expression of genes related to APP processing by regulating gene expression through its nuclear receptors. In an APP and PS1 double Tg mice, RA supplementation was associated with a significant decrease in Aβ deposition and Tau phosphorylation. Mice treated with RA also revealed decreased activation of microglia and astrocytes, attenuated neuronal degeneration, and improved spatial learning and memory in comparison to placebo [129]. 3.2.2. Experience on Humans To analyze the experience gathered with humans, we’ll categorize the studies in cohort, cross-sectional and clinical trials.

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Cohort Studies Five cross-sectional studies have been reported so far. The MoVIES project supplemented 1,059 rural, non-institutionalized elder residents of the southwestern Pennsylvania area from year 1989 to 1991. The mean age of the participants was 74.5 years. The use of nutritional supplements containing vitamin A, C, or E, β-carotene, zinc, or selenium was measured through self-report. Women and persons with higher levels of education were more often antioxidant users. Antioxidant users scored better than nonusers on several cognitive tests, but after data were controlled for age, sex, and education, there were no statistically significant relations between antioxidant use and cognitive test performance [130]. More data came from a study that investigated the association between serum antioxidant (vitamins E, C, A, carotenoids, selenium) levels and poor memory performance in an elderly, multiethnic sample of 4,809 North Americans. The data for this analysis came from the Third National Health and Nutrition Examination Survey (NHANES III) a national cross-sectional survey conducted from 1988 to 1994. The study finally concluded that decreased serum levels of vitamin E were consistently associated with increasing levels of poor memory after adjustment for age, education, income, and vascular risk factors. However, serum levels of vitamins A and C, β-carotene and selenium were not associated with decreasing memory performance [131]. The Honolulu–Asia Aging Study, is a longitudinal study of Japanese-American men living in Hawaii. Data for this study were obtained from a cohort of 3,385 men, aged 71 to 93 years followed from 1982 to 1993. Results for this trial were mixed. In the 1988 report, no protective effect was found for Alzheimer’s dementia. Among those without dementia, use of either vitamin E or C supplements alone in 1988 was associated significantly with better cognitive test performance at the 1991 to 1993 examination, and use of both vitamin E and C together had borderline significance [132]. The Chicago Health and Aging Project (CHAP) is an ongoing study that begun on 1999, following a cohort of 815, AD free volunteers over 65 years old. After a mean of 3.9 years of follow up, vitamin E intake from foods was associated with a decreased risk of developing AD after adjustment for age, education, sex, race, ApoE e4 gene, and length of follow-up. The protective association of vitamin E was only observed among the ApoE e4 negative. Surprisingly, the intake of vitamin C, β-carotene, and vitamin E from supplements was not significantly associated with risk of AD [133]. In a second report, searching for a possible explanation for the inconsistency of previous studies, the investigators examined the roles of the different tocopherol forms in the protective vitamin E association with AD and cognitive decline. They hypothesized that the protective effect was not due to α-tocopherol alone but to another tocopherol form or to a combination of tocopherol forms. Higher intakes of vitamin E and α–tocopherol were associated with a reduced incidence of AD. In separate mixed models, a slower rate of cognitive decline was associated with intakes of vitamin E, αtocopherol equivalents, and α - and γ-tocopherols, which suggests that various tocopherol forms rather than α- tocopherol alone may be important in the vitamin protective association with AD [134].

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Finally, the Nurses’ Health Study included 14,968 women, from 70–79 years of age, who participated in the study between 1995 and 2000. The study concluded that the use of specific vitamin E supplements, but not vitamin C supplements was related to modest cognitive benefits in older women. Interestingly, there was a trend for increasingly higher mean scores with increasing durations of use. Also, benefits were less consistent for women taking vitamin E alone [135]. The results of the cohort studies are not surprising. It has been well established that oxidative stress is an early event in AD. If we begin antioxidant supplementation at an advanced age we would arrive late, as the pathologic changes should had taken place and the neurodegenerative process already taking course. This is coherent with experimental evidence of the benefits of early antioxidant supplementation [126], and the benefit of prolonged antioxidant intake [135]. Prospective Studies Analyzing the prospective studies we find an even more complex perspective. The Rotterdam Study, a prospective cohort study conducted in the Netherlands followed 5,395 participants from 1990 to 1999, aged 55 years or older, free of dementia at the moment of enrolment. After adjustment for potential confounding factors, the use of high intake of vitamin C and vitamin E was associated with lower risk of AD. This relationship was most pronounced among current smokers, and did not vary by education or ApoE e4 genotype [136]. The Canadian Study of Health and Aging (CSHA) conducted a population-based, prospective 5-year investigation on 894 Canadians over 65 years of age with no evidence of dementia at baseline. Subjects reporting a combined use of vitamin E and C supplements and/or multivitamin consumption were significantly less likely to experience cognitive decline during the follow-up period. However, a reduced risk for incident dementia or AD was not observed [137]. The Washington Heights-Inwood Columbia Aging Project (WHICAP) studied 980 subjects over 65 years old, free of dementia at baseline for a mean period of 4 years. Neither intake of carotenes and vitamin C, nor vitamin E in supplemental or dietary form or in both forms, was related to a decreased risk of AD [138]. The Cache County, cross-sectional and prospective study analyzed 4,540 volunteers, 65 years or older from 1995 to 2000. The use of vitamin E and C supplements in combination was associated with reduced AD and incidence. A trend toward lower AD risk was also evident in users of vitamin E and multivitamins containing vitamin C, but no evidence of a protective effect with use of vitamin E or vitamin C supplements alone, with multivitamins alone, or with vitamin B–complex supplements [139]. Another report from the Honolulu-Asia Aging Study analyzed 2,459 men, free of dementia at the first assessment in 1991–1993 and reassessed them between 1991 and 1999. The intake of β-carotene, flavonoids, and vitamins E and C again were not associated with the risk of dementia or its subtypes [140]. A subgroup of Duke’s EPESE (Established Populations for Epidemiologic Studies of the Elderly) followed 616 volunteers from 1986 to 2000 sharing the same profile as previously

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described studies. Neither the use of vitamins C and/or E reduced the time to the onset of dementia or AD [141]. Finally, the most recent evidence came from the prospective-cohort Adult Changes in Thought (ACT) study. The study involved 2,969 participants of similar characteristics. Over a mean follow-up of 5.5 years, the use of vitamin E was not associated with the risk of developing dementia or AD. No association was found between vitamin C alone or concurrent use of vitamin C and E on either outcome [142]. Clinical Trials The Alzheimer’s disease Cooperative Study (ADCS) was the first clinical trial to address the AD burden. The researchers conducted a 2-year, double-blind, placebo-controlled, randomized, multicenter trial in patients with Alzheimer’s disease of moderate severity. A total of 341 patients received the selective monoamine oxidase inhibitor selegiline (10 mg a day), α-tocopherol (2000 IU a day), both selegiline and α-tocopherol, or placebo for two years. The primary outcome was the time to the occurrence of any of the following: death, institutionalization, loss of the ability to perform basic activities of daily living, or severe dementia. However, despite random assignment, the baseline score on the Mini–Mental State Examination was higher in the placebo group than in the other three groups, and this variable was highly predictive of the primary outcome. After Mini–Mental State base-line score adjustments, there was a significant delay in the time to the primary outcome for the patients treated with selegiline, α-tocopherol, or combination therapy days, as compared with the placebo group [143]. Later Petersen et al enrolled 769 subjects with MCI from 69 ADCS sites in the United States and Canada. Following the same controlled trial standards, subjects were randomly assigned to receive 2000 IU of vitamin E daily, 10 mg of donepezil daily (the most widely used cholinesterase inhibitor available at the time the study), or placebo for three years. The primary outcome was clinically possible or probable AD. Secondary outcomes were cognition and function. As compared with the placebo group, there were no significant differences in the probability of progression to AD in the vitamin E group or the donepezil group during the three years of treatment [144]. A large randomized trial was conducted as a part of the Age-Related Eye Disease Study (AREDS). Between 1992 and 1998, 11 clinical centers randomized 3,640 participants to receive daily antioxidants (vitamin C, 500 mg; vitamin E, 400 IU; β-carotene, 15 mg), zinc and copper (zinc, 80 mg; cupric oxide, 2 mg), antioxidants plus zinc and copper, or placebo. There was no significant effect of intake of these antioxidants on the likelihood of having cognitive impairment [145]. Finally, the group of Kang et al tested the effect of vitamin E supplementation on cognitive function using data from the Women’s Health Study (WHS), placebo-controlled trial. From 1998 to 2008, 6,377 women 65 years or older participated in the cognitive substudy. There were no differences in cognitive tests between the vitamin E and placebo groups at the first assessment (5.6 years after randomization) or at the last assessment (9.6 years of treatment). Mean cognitive change over time was also similar in the vitamin E group compared with the placebo group [146].

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Parkinson’s Disease Parkinson’s disease (PD) is the most common cause of motor disorder and the second most frequent age-related neurodegenerative disorder after AD. It affects over 1% of the population of 65 years or older, with over one million cases in the United States alone. Parkinson’s disease is a progressive disease with a median age-of-onset of 55 years. Its incidence increases markedly with age, from 20/100,000 overall to 120/100,000 at age 70 [147,148,149]. As in virtually every ND, the etiology of the disease is still unknown but probably involves a combination of genetic and environmental factors [150]. Parkinson’s disease is another appealing entity to the “Oxidative stress hypothesis” paradigm. As we will discuss in this section, it is not the biomarkers of oxidative damage in CNS tissue samples or experimental models of PD in support of the occurrence of oxidative stress that are missing. However, similar to other ND disorders, there is still lack of understanding about the triggering events of the condition and the sites of origin of ROS and RNS [151]. Clinicians have learned that it is more accurate to refer to “Parkinsonism” or “Parkinsonian syndromes,” as PD seems more of a collection of distinct neurological disorders sharing a similar clinical phenotype than a single neurological entity. There more than 40 distinct entities than can share the clinical features of PD. Clinic-pathological studies have revealed that 20% of patients with an initial PD diagnose had pathological changes suggesting other diagnose, and 5-10% of patients with Parkinsonism had the PD histological hallmarks at the moment of autopsy [1, 151]. There is still much to come to light regarding the pathogenesis of the disease. So much indeed that the question now is whether Parkinson’s disease is a single clinical entity, or the common denominator for several Parkinsonian disorders is the formation of Lewy bodies and the loss of dopaminergic neurons. Are we facing several disorders sharing a common pathogenic process that may converge at a certain point to become the same disease? Most researchers agree that the pathogenic cascade may, in fact, consist of common mechanisms. If this is the case, one may propose that similar molecular machinery is recruited, regardless of the nature of the initiating pathological factor, somehow facilitating the task of relieving the burden of these patients. Following this premise, it is possible that oxidative stress is a common event to such dopaminergic disorders [151, 152]. We will try to address some of these questions in order to identify the role of oxidative stress in PD, a main event in the neurodegenerative process, or merely an epiphenomenon in the pathophysiology of the disease.

2. Pathophysiology 2.1. Histopathology and Molecular Biology The classical pathological feature of PD is the loss of the nigrostriatal dopaminergic pathway neurons; the substantia nigra pars compacta (SNpc). The loss of SNpc neurons leads to striatal dopamine (DA) deficiency, responsible for the motor symptoms of PD.

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Characteristic clinical motor features of the disease include: resting-tremor, spasticity, postural reflex compromise and bradykinesia. Clinical symptoms of PD appear only when dopamine levels are reduced to greater than 60% that of normal [154]. Replenishment of striatal DA through the oral administration of the DA precursor levodopa (L-3,4dihydroxyphenylalanine) alleviates most of the motor symptoms [147, 153, 154]. The dopaminergic deficit though, is not sufficient to explain other signs and symptoms associated with PD. Pathologic findings in other brainstem in subcortical and cortical structures are also prominent, where widespread neuronal loss has been detected in other catecholaminergic and non-catecholaminergic nuclei. These less specific neurodegenerative processes are thought to be responsible for the complex, and non-motor symptoms including disturbances of autonomic functions and deterioration of cognition [149, 153]. Other distinguishing pathological features of PD include the accumulation of neuronal intra-cytoplasmic inclusions known as Lewy bodies (LB), found within nearly the entire characteristic affected brain areas, and gliosis. The role of LB in PD remains unclear. Hypotheses are very distinct, ranging from a possible contribution in cell death, to nonsignificant relevance. Others postulate that their occurrence might indicate that the cell is evading death by successfully rendering misfolded proteins harmless. Lewy bodies are particularly rich in aggregated α-synuclein, but also contain several other proteins, including components of the ubiqiuitin–proteasome system and molecular chaperones, and lipids. The proportion of neurons containing LB in PD remains relatively constant at 4% regardless of disease stage. It has therefore been suggested that LB are continuously formed during the course of the disease and disappear when the affected neuron dies [149, 151]. Alpha-Synuclein is normally abundant in nerve terminals where it’s involved in synaptic function. Alpha-Synuclein is lipid-associated under physiological conditions but tends to aggregate in the lipid-free state into higher molecular weight oligomers. Formation of these aggregates has been directly linked to neurodegeneration in PD. Two mutations in the αsynuclein gene (A30P and A53T) have been associated with familial PD, but the biochemical consequences of these mutations are not completely understood. What is known is that during normal aging and in PD, levels of natively folded α-synuclein increase in the neuronal cytoplasm [155]. Parkinson’s disease can be classified as primary when it’s due to hereditary or idiopathic causes or secondary when it has an identifiable underlying pathology. Parkinson’s disease is a sporadic disease in 95% of the cases; however several genes that lead to a hereditary parkinsonian disorder have been identified in the last few years. Rarely occurring genetic defects include mutations in genes such as α-synuclein, parkin, DJ1, PINK1, and LRRK2. These mutations are transmitted as either dominant or recessive Mendelian traits. Clinically both, sporadic and familial forms of PD are almost indistinguishable sharing the same basic biochemical hallmark of a profound dopaminergic deficit [151, 152]. Considering the biochemical function of these genes and mutations, three main, interrelated, underlying pathogenic pathways can be identified. These are: altered protein quality control; oxidative stress and mitochondrial dysfunction; and disturbed kinase activity. The common pathway to these pathologic processes is cellular death, executed by exitotoxicity, apoptosis and autophagy, being apparently facilitated by the pro-oxidant, neuroinflammatory progression [152].

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Prior to the last 5 years, most of the generated evidence regarding the etiology and pathogenesis of PD was derived from postmortem tissue of animals models exposed to the neurotoxic compound 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Exposure to MPTP induces a dopaminergic neurodegeneration that causes a syndrome that mimics PD, also common to humans. The more recent discovery that mutations in the gene for αsynuclein can cause an inherited form of PD changed the field completely. Several genes involved in the pathogenesis of PD have been described to these days. As in AD, these rare PD genes appear to operate through common molecular pathways. This has led to the development of novel animal models for the study the disease [147]. We will now revise the role of oxidative stress in the disease, gathered thanks to this previous knowledge.

2.2. Oxidative Stress in Parkinson’s Disease As in Alzheimer’s disease, there is growing evidence regarding the importance of oxidative stress in the pathogenesis of PD. However, there is still much discussion whether oxidative stress is the primary insult or a secondary event. We will once again explore the source of ROS in these neurodegenerative processes, and their relation to the antioxidant endogenous defense. Finally, we’ll review the most relevant oxidative stress-derived biological markers of the disease. 2.2.1. Sources of Reactive Oxygen Species and Antioxidant Defense Evidence now exists to support a role for alterations in mitochondrial form and function, as well as increased oxidative stress in the pathogenesis of PD [153]. Factors contributing to the increased oxidative stress in PD include: high basal levels of aerobic activity in the brain, auto-oxidation of DA, its precursors, and metabolites, to form quinones and semiquinones capable of adducting protein sulfhydryl groups, including GSH, and increased iron levels in the SNpc contributing to Fenton chemistry through the reduction of hydrogen peroxide (H2O2) to produce the highly reactive hydroxyl radical (OH•). Histological findings in postmortem brains from PD patients, confirm high levels of oxidative stress in the SNpc, as revealed by increased iron concentrations, decreased levels of GSH, increased lipid peroxidation, and DNA and protein oxidation [148]. However, the earliest reported event of oxidative stress in PD is depletion of GSH. The GSH depletion precedes mitochondrial damage and dopamine (DA) loss and the degree of its loss correlates with the disease’s severity [156]. Proteasome inhibition is another phenomenon of main relevancy affecting neuronal viability in PD. Proteasomal inhibition in animal models results in most of the biochemical hallmarks and selective brain damage of PD [157]. Proteasome inhibition has also been associated to altered mitochondrial lysosomal-mediated degradation and homeostasis, and in combination with alterations in GSH metabolism [158, 159]. A growing number of clinical and experimental evidence implicate mitochondrial dysfunction as a fundamental aspect of the increased oxidative stress in the pathogenesis of PD [150]. Mitochondria isolated from PD patients displays reduced complex I activity [160].

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Impairment of the respiratory chain may lead to increased ROS generation, leading to more oxidative stress and macromolecular damage and selective death of SNpc of PD patients. A complicated interplay occurs between mitochondria and other cellular machinery that dramatically affects cellular survival [148, 153]. Mitochondrial dysfunction also leads to increased DA oxidation in its reservoirs, increasing the damage by liberation of the oxidized neurotransmitter. The oxidative stressinflammation cycle can also lead to microglial activation. Microglia activation has been associated with upregulation of iNOS and NADPH oxidase, leading to increased nitrous oxide and superoxide anion production respectively. In this sense, augmented protein nitration has been observed in Lewy bodies’ together with more lipid peroxidation and DNA strand breakage in areas affected by PD [148]. Accordingly, mutant mice defective in NADPH-oxidase exhibit less SNpc dopaminergic neuronal loss and protein oxidation than their wild type littermates after MPTP injections [161]. Also, pharmacologic inhibition of NOS significantly protects MPTP-injected mice against injury to the SNpc [162]. However, there is still much discussion whether oxidative stress is a primary or secondary event to the genetic injury. Several PD-associated genes have been found to influence the balance of mitochondrial fission and fusion, affecting the maintenance of dynamic networks of these organelle’s tubular structures [153]. Genetic studies additionally have linked mitochondrial dysfunction with several genes expressing their phenotype in PD such as α-synuclein, parkin, DJ-1, PINK1, and LRRK2 [163]. 2.2.2. Oxidative Stress and Genopathies Clinically, both sporadic and inherited forms of PD are virtually indistinguishable. Sharing the biochemical stamp of a profound DA deficit in the SNpc, they have provided with deeper understanding of PD’s neurodegenerative mechanisms [151]. Mutations to the autosomal dominant LRRK2 gene represent the most frequent cause of hereditary PD, affecting up to 7% of all PD patients in Europe and up to 40% in Ashkenazi Jews and North African Arabs [152, 163]. The LRRK2 gene possesses two catalytic domains: a Ras/GTPase super family, and as serine-threonine kinase. Disinhibited kinase activity associated to such mutations appears to induce a progressive reduction in neurite length and branching, both in vitro and in vivo. The PD-associated LRRK2 mutations additionally harbor prominent phospho-tau-positive inclusions with lysosomal characteristics and ultimately undergo apoptosis [164]. The PINK1 gene codifies for a serine-threonine kinase with a mitochondrial-targeting motif at its N-terminal end [165]. This protein has been localized in mitochondrial fractions, where it is thought to play a role in normal cellular physiology, protecting mitochondria from oxidative stress and apoptosis [148]. The loss of PINK1 function in mice does not cause major ultrastructural changes in their mitochondria, but instead leads to functional deficits in a dopaminergic circuit and age-specific manner with increased sensitivity to oxidative stress [150]. Mutations of DJ-1 have also been related to the pathogenesis of PD. This putative antioxidant expresses ubiquitously and localizes to the mitochondrial matrix where it is capable of reducing ROS formation directly and stabilizing the major antioxidant gene

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regulating the transcription factor Nrf2 [148]. Over expression of DJ-1 confers enhanced protection against oxidative stress, while the genetic DJ-1 knockout has an increased sensibility to ROS [166, 167].

2.3. Biological Markers of Parkinson’s Disease The same general guidelines as in Alzheimer’s disease apply for PD. The brain is a highmetabolism organ, rich in fatty acids. Damage to virtually all macromolecules is ubiquitously seen in autopsy materials obtained from PD patients and evidenced by increased lipid peroxidation, protein carbonyl modifications, and DNA oxidation. Nitration and nitrosylation of proteins, particularly α-synuclein and parkin has been documented in PD [151]. Common deletions of mitochondrial DNA have been reported in DA neurons of the SNpc [168]. These findings have proved useful in the understanding of the mechanisms of the disease. However, the challenge now is to develop biomarkers useful in everyday practice. In this regard, several researchers have validated pathologic features of PD in peripheral samples such as blood and CSF. The diagnosis of sporadic PD has remained basically clinical. Accuracy of clinical diagnoses of PD can reach 90% within experienced specialists [169]. The development of biomarkers for PD for identifying individuals in risk has been highly desired as in any other ND, particularly for the important implications of an early, accurate diagnosis and consequential treatment, as to prevent, and monitorize the progression of the disease. Furthermore, biomarkers could give further insight of the mechanisms of the disease for a better understanding of PD, but also of atypical Parkinsonian disorders, generally unresponsive to current treatments [170]. Markers of oxidative stress in peripheral blood have been recently determined in PD patients and age-matched controls. Patients with PD had significantly higher SOD activity and increased lipid peroxidation products (TBARS content) [171]. In a prospective population-based cohort study among 4,695 participants aged 55 years and older, with an average of 9.4 years of follow-up, serum levels of uric acid were also associated with a significantly decreased risk of Parkinson disease [172]. Oxidative damage to proteins is also significantly increased. On the other hand, levels of 8-OHG in serum and urine have not revealed to be trust-worthy markers of the disease. Neither has the GSH/GSSG relation, who has not significantly shown differences between PD patients and age-matched controls [170, 171].

3. Antioxidants in Parkinson’s Disease 3.1. Antioxidant Properties of Current Treatments Several therapeutic options are available or being tested for the treatment of PD. However, none except the MAO-B inhibitor rasagiline and coenzyme Q10 have been capable of modifying the course of the disease [152]. Most approaches have been targeted to cell death mechanisms, not the initial events of the disease. Monoamine oxidase (MAO) appears

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as a relevant source of ROS in PD. Pharmacologic MAO-B inhibition (iMAO-B) with deprenyl, is a widely used clinical approach. The iMAO-B rasgiline has proved antiparkinson activity in classical MPTP neurotoxin mice models, where it prevents the degeneration of SNpc dopaminergic neurons. However, there is evidence that the neuroprotective activity of rasagiline is independent of MAO-B, and yet its actual mechanism is yet to be elucidated [173]. Recent reports from clinical trials have confirmed the protective role of new iMAO-B, suggesting that MAO-B could be a relevant source of ROS in PD [154]. 3.2. Promising New Antioxidants Compounds Another important pharmacologic therapy being studied to diminish oxidative stress in PD is iron chelation. The potent iron chelator VK-28 is capable of crossing the BBB. It has proven neurotoxin protective activity in rats. The use of iron chelators in combination with iMAO has yielded promising results [154, 173]. Further findings came from the ironneuromelanin relationship. Neuromelanin binds iron and accumulates during the aging process. In PD, dopaminergic neurons containing neuromelanin selectively degenerate. Neuromelanin blocks hydroxyl radical production by Fenton’s reaction, in a dose-dependent manner. Neuromelanin also inhibited the iron-mediated oxidation of ascorbic acid, thus sparing this major antioxidant molecule in the brain. Blockade of iron into a stable ironneuromelanin complex prevented DA oxidation, inhibiting the formation of neurotoxic DA quinones. This process occurs intracellularly during aging and PD, thus providing evidence of the neuroprotective properties of neuromelanin [174]. There is also increasing interest in the micronutrient coenzyme Q10 (CoQ10) to treat neurodegenerative processes. CoQ10 is a fundamental component of the mitochondrial electron transport chain, accepting electrons from complexes I and II. It serves an important antioxidant role in both, mitochondria and lipid membranes through its interactions with αtocopherol. Coenzyme Q10 is also an obligatory co-factor for mitochondrial uncoupling proteins. Activation of these proteins reduces mitochondrial uncoupling in the SNpc of primates, providing neuroprotection against MPTP toxicity [110]. A multicenter, randomized, placebo-controlled clinical trial analyzed the neuroprotective role of CoQ10. Eighty patients, diagnosed with PD in the previous five years were randomized to CoQ10 at doses of 300-1200 mg/d or placebo. They were then followed up for up to 16 months. The main endpoint to measure was tremor score of the United Parkinson’s Disease Rating Scale (UPDRS). Coenzyme Q10 was well tolerated under the studied dosage and associated with significantly better UPDRS scores [175]. Even though the results are promising, further larger studies, with a longer follow-up time will be needed to evaluate the actual benefit of CoQ10 in delaying the progression of PD. Other antioxidant substances under current evaluation and possible antiparkinsonian properties are ginko biloba, L-carnitine, cannabis, estrogen and nicotinamide. The potent antioxidant properties of the polyphenols found in green tea are also being tested [154]. In large Asian population cohort studies, neither green tea drinking, nor diet was related to PD risk [176]. The putative antioxidant role of cannabinoids has been previously questioned. However, it has been recently demonstrated that the cannabinoid CP55,940 significantly

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protects and rescues D. melanogaster against a neurotoxic injury through a receptorindependent mechanism [177].

3.3. Antioxidant Supplementation 3.3.1. Glutathione Glutathione levels can be raised by slowing its degradation, or by supplementation with GSH itself or analogs. As GSH does not easily cross the BBB due to its charged cysteine-SH group, GSH esters have been explored as an alternative. In vivo and in vitro studies have demonstrated that the GSH precursor glutamyl cisteine ester (GCEE) and glutathione ethyl ester (GEE) are capable of elevating intracellular GSH levels, and provide protection to dopaminergic neurons against MPTP induced neurotoxicity [154, 178]. In the only published clinical trial, the effects of GSH supplementation were studied in nine patients with early, untreated PD. Glutathione was administered intravenous, 600 mg twice daily, for 30 days, in an open label fashion. The drug was then discontinued and a follow-up examination carriedout at 1-month interval for 2-4 months. Thereafter, the patients were treated with carbidopalevodopa. Clinical disability was assessed by different rating scales at baseline, and at 1month intervals for 4-6 months. All patients improved significantly after GSH therapy, with a 42% decline in disability. Once GSH was stopped the therapeutic effect lasted for 2-4 months [179]. 3.3.2. Antioxidant Vitamin Supplementation Only a few clinical trials with antioxidant vitamin supplementation have been directed with limited success. A combination of high doses of α-tocopherol and ascorbate were administered to patients with early PD in an open-labeled pilot study. Patients were allowed the concomitant administration of amantadine and anticholinergics, but not levodopa. The primary endpoint was time until patients needed treatment with levodopa or a dopaminergic agonist. The time when levodopa became necessary was extended by 2.5 years in the group under antioxidant supplementation [180]. In 1987 the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) was initiated to examine the eventual benefits of the iMAO-B deperenyl and α-tocopherol in slowing the progression of PD. After 14 months of observation, deperenyl significantly delayed the necessity of levodopa. The effect was largely sustained after 8.2 years of follow-up. However, supplementation with αtocopherol did not show any benefits in delaying the progression of PD [181].

4. Conclusions and Perspectives Neurodegenerative diseases and Alzheimer’s disease in particular have changed the epidemiological situation, as we knew it. As the principal risk factor for developing AD is aging, the disease presents a heavy social and economic burden to our society.

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With a poorly understood pathogenesis, the task to develop novel therapeutic approaches to prevent or delay the progression of the disease, or even alleviate our patient’s symptoms, has proved a frustrating endeavor. According to the current evidence about the underlying mechanisms of the disease we can say, at least, that oxidative stress is a mayor initial event (if not the most relevant), in the triggering of the neurodegenerative process. In this regard, several sources of ROS have been revealed in the recent years. Especially important, has been the identification of Aβ peptides as ROS producing agents per se and also participating as pro-oxidative mediators in pathologic events such as mitochondrial dysfunction. However, the presence and relevancy of other pro-oxidative cofactors, and endogenous antioxidants is yet to be elucidated. Consequent to a major role for oxidative stress in the pathogenesis of neurodegenerative processes, human observational epidemiology studies have been in general consistent with the hypothesis of an inverse relationship between antioxidant defense, cognitive function and ultimately the risk of developing AD. In spite of disappointing results of clinical trials, there are several caveats to these findings. Virtually all of them omit important information such as drug-level monitoring and and/or biomarkers to support the therapeutic effect of the tested treatment. But also, in all of them the intervention or the follow-up began at 65 years of age or older. We know from genetic, dietary and pharmacologic approaches, mainly derived from animal models, but also human, that the most important benefits have been obtained when the diagnosis of AD is early and the antioxidant supplementation is established at a young age, before deposition of Aβ plaques. This suggests that longer and earlier antioxidant supplementation should be stated to address the problem. But then again, we must face the increased cardiovascular risk that has been recently associated to antioxidant supplementation with free-radical scavengers such as tocopherol and retinoic acid. In other words, what is it that represents such a benefit to our cognition but can be so adverse to our cardiovascular status? In this regard, the increased life expectancy associated to the use of dietary supplements such as green tea, curcumin, or even better, full mediterranean diet sounds very promising to come in aid of neurodegenerative diseases. For Parkinson’s disease, on the other hand, we glimpse a less optimistic panorama. Only a few current therapeutic interventions are aimed to alter the progression of the disease. On top of that, our understanding of its pathogenesis is very limited. However great is evidence that was first obtained from neurotoxic models, and then from the description and reproduction of genetic defects associated to familiar PD. Many of these anomalies are neurochemically indistinguishable from the sporadic disorder. From all gathered evidence, Parkinson’s disease displays as several disorders converging into a unifying neurodegenerative process with its own recognizable clinical features. Also, oxidative stress is also an early event in the pathogenesis of PD. This raises the hope that is possible to intervene with antioxidant supplements early in the progression of the disease, to provide neuroprotection significant in delaying the progression of PD. However the results of (very few) clinical trials testing this sort of interventions have not been quite encouraging. Are we facing the same limitations AD clinical research has had to cope with? Whatever the answer to these questions it’s not difficult to uphold the thesis that oxidative stress is a main event of the neurodegenerative process, and as such should be further explored.

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In: Oxidative Stress and Antioxidants Editor: Ramon Rodrigo

ISBN: 978-1-60741-554-1 © 2009 Nova Science Publishers, Inc.

Chapter XI

Glaucoma Leonidas Traipe1, Rodrigo Castillo2 and Ramón Rodrigo2 1

Ophtalmologic Foundation “Los Andes”; Santiago; Chile 2 Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile. Supported by FONDECYT, grant 1070948

Abstract Glaucoma constitutes an increasingly serious public health problem, moreover in developed countries and is an important cause of blindness after cataracts. It is an optic neuropathy that implies loss of retinal ganglion cells, including their axons, and a major tissue remodeling, especially in the optic nerve head. Although increased intraocular pressure is a major risk factor for glaucomatous optic neuropathy, there is little doubt that other factors such as ocular blood flow play a role as well. Mechanisms leading to glaucomatous optic neuropathy are not yet clearly understood. There is, however, increasing evidence that both activation of glial cells and oxidative stress in the axons play an important role. The involvement of reactive oxygen species (ROS) in the pathogenesis of glaucoma is supported by various experimental findings, including: (i) resistance to aqueous humor outflow is increased by hydrogen peroxide by inducing trabecular meshwork (TM) degeneration; (ii) TM possesses remarkable antioxidant potential, mainly explained by superoxide dismutase and catalase activities and glutathione pathways, all that is found decreased in glaucoma patients; and (iii) intraocular-pressure increase and severity of visual-field defects in glaucoma patients paralleled by the amount of oxidative damage of DNA affecting TM. Vascular alterations, which are often associated with glaucoma, could contribute to the generation of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal cells, appears to be involved in the neuronal cell death affecting the optic nerve in glaucoma. Despite the major pathogenic role of ROS in the pathophysiology of glaucoma, clinical trials testing the efficacy of antioxidant drugs for its management are still lacking.

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1. Introduction Glaucoma is an insidiously progressive optic neuropathy which affects nearly 90 million people worldwide, and is the leading cause of irreversible blindness after cataracts. It is a neurodegenerative disorder of aging and occurs with increasing prevalence after the age of 40 [1] . Although data are incomplete and not definitive, primary open angle glaucoma (POAG) has been estimated to affect about 2% of the population of Italy [2]. It is estimated that only 40% of glaucoma-affected subjects undergo adequate therapy, the absence of early severe symptoms often contribute to delay the diagnosis [3]. Indeed, POAG is a chronicdegenerative disease in which the clinical manifestations are preceded by a long latency. This is of major relevance due to once the damage affecting visual capacity arises; it is no longer reversible [4]. There are important risk factors for POAG such as presence of elevated intraocular pressure as yet in the absence of visual-field damage (prevalence 6%) [5]. Advanced age represents a fundamental risk factor for this disease [6]. Increasing experimental evidence indicates that oxidative stress plays a major role in the pathogenesis of the main cause of irreversible blindness, the degenerative primary open angle glaucoma (POAG) [7, 8]. Indeed, tissues from the trabecular meshwork (TM) in patients with primary open angle glaucoma have been reported to contain a high amount of metabolic products of lipid peroxidation suggesting a role for oxidative stress in the pathophysiology of this disease [9]. Recently, some experimental models have associated the elevation of intraocular pressure with the occurrence of oxidative retinal damage thus giving a clue for the use of antioxidant administration in order to attenuate the progression of the structural and functional irreversible damage. Despite the major pathogenic role of ROS in the pathophysiology of glaucoma, clinical trials testing the efficacy of antioxidant drugs for its management are still lacking.

2. Pathophysiology of Glaucoma 2.1. General Concepts Glaucoma is an optic neuropathy characterized by a specific structural alteration of the head of the optic nerve accompanied by a progressive damage to the visual field. Although increased intraocular pressure (IOP) is a major risk factor for POAG, other concomitant factors affecting the eye play important roles including increased glutamate levels [10], alterations in nitric oxide (NO) metabolism [11], vascular alterations [12] and oxidative damage caused by ROS [13]. 2.1.1. Outflow Pathway The conventional outflow pathway consists of trabecular lamellae covered with human trabecular meshwork (TM) cells, in front of a resistor consisting of juxtacanalicular human TM cells and the inner wall of Schlemm’s canal. The outermost juxtacanalicular or cribriform region has no collagenous beams, but rather several cell layers which some authors claim to be immersed in loose extracellular material/matrix [14].

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The main resistance to the aqueous humour outflow is located in the TM directly underneath the inner wall of Schlemm’s canal [15, 16]. Ultrastructural changes in glaucomatous TM are similar to, but much more intense than, those observed in the normal TM in the elderly [17]. These changes include the thickening of basal membranes and trabecular beams, their enlargement or collapse, partial loss of endothelial cells and the accumulation of materials such as pigment granules and calcium precipitates [18], central nucleus changes such as an increase in electrodense plaques and collagen, and loss of endothelial cells [18]. Furthermore, outflow resistance increases with age [20]. Thus, the increase in oxidative DNA damage in the cellular component of the TM could directly affect regulation of the extracellular matrix structure and the associated regulation of intraocular pressure, leading to the clinical onset of glaucoma [21, 22] When the intraocular pressure (IOP) exceeds the pressure in the episcleral venous plexus, the endothelial cells that line the lumen of Schlemm’s canal (SCEs) form ‘‘giant vacuoles’’ that facilitate the aqueous outflow in this condition [23]. Similarly, SCEs discourage the reflux of blood by preventing the formation of giant vacuoles when the episcleral venous pressure exceeds the IOP [24]. Indeed, these endothelial cells release vasoactive cytokines and other factors able to increase the permeability of the endothelial barrier of the Schlemm’s canal. Alterations in these cytokines may not allow sufficient flow through Schlemm’s canal and, consequently, the IOP may rise to abnormal levels [25]. This interpretation of glaucoma pathophysiology is in agreement with the view that increased intraocular pressure (IOP) is secondary to a decline in trabecular meshwork cellularity [26]. Lutjen-Drecoll [19] has recently claimed that ‘‘common factors are involved in the pathogenesis of both the TM and the optic nerve changes’’. The common denominator involved in the cellular alterations of the TM structure and optic nerve damage is on one hand the oxidative stress, and on the other the vascular damage described both in glaucoma [27] and aging. 2.1.2. Retinal Glutamate/Glutamine Cycle Activity Glutamate is the main excitatory neurotransmitter in the retina, but it is toxic when present in elevated concentrations. Retinal tissue is in fact an established paradigm for glutamate neurotoxicity for several reasons: (i) insult leads to accumulation of relatively high levels of glutamate in the extracellular fluid [28]; (ii) administration of glutamate leads to neuronal cell death [29]; and (iii) glutamate receptor antagonists can protect against neuronal degeneration [30]. Thus, an appropriate clearance of synaptic glutamate is required for the normal function of retinal excitatory synapses and for the prevention of neurotoxicity. Glial cells, mainly astrocytes and Müller glia, surround glutamatergic synapses and express glutamate transporters and the glutamate-metabolizing enzyme glutamine synthetase [31, 32]. Glutamate is transported into glial cells and amidated by glutamine synthetase to the nontoxic aminoacid glutamine. Glutamine is then released by the glial cells and taken up by neurons, where it is hydrolyzed by glutaminase to form glutamate again, completing the retinal glutamate/glutamine cycle [33, 34]. In this way, the neurotransmitter pool is replenished and glutamate neurotoxicity is prevented. Glutamatergic injury has been proposed to contribute to the death of retinal ganglion cells in glaucoma. This hypothesis is supported by the demonstration that vitreal glutamate is

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elevated in glaucomatous dogs [35, 36] and quail with congenital glaucoma [37]. In addition, high glutamine levels have been found in retinal Müller cells of glaucomatous rat eyes [38]. In contrast, other authors showed no significant elevation of glutamate in the vitreous humor of patients with glaucoma [39], or in rats [40] and monkeys with anatomic and functional damage from experimental glaucoma [41]. In any case, it seems limited to assume that high levels of glutamate in the vitreous are a necessary condition for excitotoxicity to be involved in glaucomatous neuropathy. The local concentration of glutamate at the membrane receptors of ganglion cells is the important issue for toxicity. This could be very different from the level in samples of vitreous humor. Vitreous humor must be removed for experimental measurement by a process that inevitably disturbs its state before removal. These manipulations could themselves alter the measured glutamate concentration. With the only exception of glutamine synthetase, the changes of glutamate/glutamine cycle parameters were transitory. Although this issue has no ready explanation, it is possible that these changes were provoked by a reversible injury to Müller or other retinal cells more than with cell death. The changes in glutamate recycling preceded functional and histological alterations induced by ocular hypertension [42]. Therefore, it is tempting to speculate that the changes in glutamate/glutamine cycle activity could be a causal factor in ocular hypertensioninduced neuropathy. Furthermore, it seems possible that the increase in synaptic levels of glutamate could represent an initial (and probably reversible) insult responsible for the initiation of damage that is followed by a slower secondary degeneration that ultimately results in cell death. It has been previously described that decreased in the retinal antioxidant defense system potential at 6 weeks of treatment with hyaluronic acid [13, 44], and other authors have postulated that excessive levels of NO may contribute to this optic neuropathy [45]. With respect to these data, the hypothesis that the retinal damage induced by ocular hypertension may result, at least in part, from oxidative stress induced by a glutamate/mediated pathway, as shown in other neuronal injury models [46, 47]. 2.1.3. Nitric Oxide and Neurotoxicity Nitric oxide is an important messenger intra and extra molecular implicated in vasodilatation, contractility, neurotransmission, neurotoxicity and inflammation. Nitric oxide has a demonstrate role in many neurodegenerative diseases like: glaucoma, Alzheimer disease, multiple sclerosis and cerebral-cardio-vascular diseases [48]. In this point, Ferreira et al. [49] analyzed the implication of NO and nitrosative stress in the pathophysiological mechanism of glaucoma, emphasizing the importance of biochemical markers in the aqueous humor to evaluate the progression of the glaucomatous optic neuropathy. A significant increase in the NO concentration, malondialdehyde levels, and a significant decrease of the total antioxidant status were detected in patients having trabeculectomy. This study is in agreement with other findings in some experimental models such as retinal ganglionar cell line [50] and rats [51]. Recently, it has been proposed that the increase in NO formation in the aqueous humor in those patients with glaucoma, in the presence of oxidants, should induce the formation of peroxynitrite and it is very possible that nitrosative stress is induced as a result of this. This point is an essential goal to be handled when it comes to future experimentation within this line of research [52]. In this view, besides of evaluating the sources of NO in anterior chamber, the trabecular distribution of NOS suggests an important

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role of nitric oxide in the future therapies for the glaucoma. The increase of nitric oxide produces vasodilation and improves contractility in the TM; the final effect being the decrease of intraocular pressure and on the other hand the contra-apoptotic effect giving neuroprotection [53].

2.2. Role of Oxidative Stress in the Development of Glaucoma 2.2.1. Defenses Against Free Radicals and the Eye Both vitamin C and glutathione operate in fluid both outside the cell and within the cell [54], whereas vitamin E prevents endogenous mitochondrial production of ROS [55]. This may be important in maintaining cellular homeostasis, which is relevant to counteract the mechanism leading to the development of POAG [56]. Indeed, vitamin E prevents apoptosis during hypoxia and oxygen reperfusion [57], and it may reduce apoptosis by means other than antioxidation [58]. Ascorbic acid is thought to be a primary substrate in ocular protection because of its high concentration in the eye. Within the cell, vitamin C helps to protect membrane lipids from peroxidation by recycling vitamin E [59]. It is present at high concentrations in vitreous humor [60], cornea [61] and tear film [62]. One of ascorbate’s presumed functions is to protect the lens and retina from the damaging effects of ultraviolet radiation [63]. In addition, it exerts a filter-like function against UV radiation in both the central corneal epithelium and aqueous humor [64] and reacts with O2 to form H2O2. The direct correlation between the concentrations of ascorbic acid and H2O2 in aqueous humour suggests that ascorbic acid is the primary source of H2O2 in this fluid [65], although it has been claimed that H2O2 levels can be overestimated [66]. A high level of ascorbic acid is necessary to maintain oxidative balance in the aqueous humor, while vitamin E deficiency increases H2O2 levels [55]. Unfortunately, these processes are not able to eliminate free radicals completely, and, if oxidative stress is very severe, it may ultimately cause cell death [67]. On the other hand, the GSH redox system is believed to protect ocular tissues from the damage induced by low H2O2 concentrations, whereas catalase is thought to protect ocular tissues from the damage induced by higher H2O2 concentrations [68, 69]. A decline of catalase activity with age has been observed in the iris and in the corneal endothelium of rabbits [70, 71]. Both glutathione and ascorbate have been detected in aqueous humour [72, 73]. These antioxidants seem to play a particularly important role in glaucomatous disease. Indeed, patients with glaucoma exhibit low levels of circulating glutathione, suggesting a general compromise of antioxidant defenses [74]. Moreover, genetic polymorphisms have been detected for GSH transferase isoenzyme, and the GSTM1-null genotype has been found to be significantly more common in patients with POAG than in controls [75]. On the basis of the currently available literature, free radicals seem to play an important role in the pathogenesis of ocular diseases. Indeed, ROS are a cause of cataract [10, 76], are implicated in age-related macular degeneration [77, 78], and also may play a significant role in the pathophysiology of glaucoma [49, 79]. Vascular damage and neuronal cell death associated with glaucoma and role of oxidative stress is show in figure 11-1.

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Figure 11-1. Intraocular pressure increase in glaucoma progression and relationship with oxidativerelated degenerative processes affecting TM. ONOO-: peroxynitrite; TM: Trabecular meshwork; IOP: intraocular pressure.

2.2.2. Oxidative Stress and Intraocular Pressure Elevation The pathogenic role of oxidative stress in increasing IOP by reducing aqueous outflow facility is supported by various experimental studies performed in vitro and in vivo. In vitro treatment of human TM cells with H2O2 alters cellular adhesion and integrity [80]. In an animal model of study, perfusion of TM with peroxide has shown to reduce aqueous humor drainage from the anterior chamber of the calf’s eye [81]. In humans, oxidative DNA damage has been reported to be significantly higher in the TM cells of glaucoma patients than in those of age-matched controls [75]. Further studies demonstrated abundant oxidative nucleotide modification (8-OH-dG) levels in human TM to be significantly correlated to the increase in IOP and to visual field damage [79]. Further evidence suggests that patients with POAG exert mitochondrial abnormalities implicating that mitochondrial dysfunction is most probably a consequence of oxidative stress [82]. Free radicals, contained in the aqueous humor, contribute to pathogenic alterations in the TM [20]. It was shown increased resistance to the outflow of aqueous humor of calf is a result of TM cytoskeletal rearrangements and cellular loss, in the presence of increased levels of H2O2. The damage caused by prooxidants to the aqueous humor outflow system accounts for reports signaling that radiologists suffer from ocular hypertension more frequently [83]. At the molecular level, human TM endothelium has been reported to be an enriched site of NO synthesis. Nitric oxide can interact with oxygen or metals, such as copper or iron, to modulate outflow resistance of the TM [84].

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2.2.3. Oxidative Stress and Glaucomatous Optic Neuropathy Glaucomatous optic neuropathy (GON) is the optic nerve damage secondary to progressive and chronic enhancement of IOP in glaucoma patients. However, not all glaucoma patients suffer from elevated IOP, along with the observation that the majority of these patients show signs of reduced ocular blood flow as well as ischemic signs in the eye (e.g., upregulation of the ischemia inducible factor 1-α) [85] suggesting that hemodynamic factors are involved in this process. Glaucoma patients have reduced ocular blood flow, what has a predictive power for the progression of GON [86]. On the other hand, a blood flow reduction by atherosclerosis, multiple sclerosis, or other similar ischemic diseases, increases the risk for GON insignificantly. The solution of this seemingly paradox observation is simply the fact that it is not a stable reduction in ocular blood flow but rather the instability in blood flow that may lead to GON [87]. Such an instability in ocular blood flow leads to a repeated mild reperfusion injury [88]. This hypothesis is supported by the observation that IOP fluctuation is more damaging than a stable increase in IOP [89, 90] and by observations that patients that progress despite a normalized IOP suffer from a disturbed autoregulation [91]. The main cause for this insufficient autoregulation is a primary vascular dysregulation syndrome [92]. The term dysregulation simply means that blood flow is not properly adapted to this need. Dysregulative mechanisms can lead to an over- or underperfusion. A steady overperfusion may be less critical for long-term damage. A constant underperfusion, however, can lead to some tissue atrophy or in extreme situations to infarction. Unstable perfusion (underperfusion followed by reperfusion) leads to oxidative stress [93]. As described previously, oxidative stress occurs under a condition of high energy consumption, light exposure, or age-dependent decline of coping capacity to deal with free radicals. In glaucoma, an additional major factor is most likely a repeated mild reperfusion injury. This hypothesis is supported by observations on circulating lymphocytes of human glaucoma patients. Activated astrocytes express MHC-II capable of communicating with lymphocytes and lymphocytes also communicate with the capillary endothelial cells during reperfusion [94]. Indeed, the lymphocytes of glaucoma eyes express neural thread protein [95], indicating axonal damage, but they also reveal upregulation of p53 and of proteosome 20 S subunits, along with an over-expression of metalloproteinase-9. Moreover, glaucoma patients present higher plasma lipid peroxidation products concentration in comparison to controls [96]. These reports together with observations of increased DNA breaks in circulating lymphocytes [97] support the assumption of the occurrence of oxidative stress in glaucoma patients [98]. On the other hand, in the optic nerve head, blood flow is particularly unstable as a result of mechanical stress and an insufficient blood--brain barrier giving access of vasoactive substances to the pericytes and smooth muscle cells [89] . In addition, there is very high energy consumption in the axons of the optic nerve head due to the lack of myelin sheaths and therefore a high concentration of mitochondria in this area. Therefore, it is very important to describe the role of the pathological process derived from ischemia-reperfusion cycle in eyes, mainly the retina cells. During reperfusion the main source of free radicals in cells lacking xanthine oxidase (all neural cells) are the mitochondria [ 99]. Whereas oxidative stress damages all types of molecules and thereby reduces the probability of cellular survival,

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the question arises as to why in glaucoma specifically the retinal ganglion cells and their axons die by apoptosis. Reperfusion injury in glaucoma patients, particularly in the optic nerve head, is very mild but occurs repeatedly. The assumption of reperfusion injury being involved in the pathogenesis also explains why sleep apnea [100] or reversible shock--like states [101] can lead to GON. Reactive oxygen species damage biomolecules such as proteins or lipids of plasma membranes. The damage to the cells, in turn, causes the release of more free radicals. In prolonged ischemia hypoxanthine is formed as a result of the breakdown of ATP and the enzyme xanthine dehydrogenase is converted to xanthine oxidase. This also results in molecular oxygen being converted to the highly reactive superoxide and hydroxyl radicals, further resulting in tissue damage [102]. The nerve cells of CNS, however, lack xanthine oxidase (with the exception of blood vessels). In the central nervous system, ROS formation therefore does not occur via xanthine oxidase. The major source of oxidative stress in reperfusion stems from the mitochondria which are very crowded in the optic nerve head due to high energy consumption in these nerve fibers lacking myelin sheaths [103] . The role of oxidative stress is further supported by findings of weaker antioxidant defense systems in POAG patients. 2.2.4. Experimental Models of Glaucoma and Oxidative Stress Rats are becoming an increasingly used model system for understanding the mechanisms of optic nerve injury in POAG. Although the anatomy of the rat optic nerve head is different from that of humans, the ultrastructural relationships between astrocytes and axons are quite similar, making likely that cellular processes of axonal damage in these models will be relevant to human glaucoma [104]. All of these models rely on elevating IOP, a major risk factor for glaucoma. Methods that produce increased resistance to aqueous humor outflow at the anterior chamber angle, specifically hypertonic saline injection of aqueous outflow pathways and laser treatment of the limbal tissues, appear to produce a specific regional pattern of injury that may have a particular relevance to understand the regional injury in human glaucoma. Increased pressure and its fluctuations are a characteristic of such models and the rodent optic nerve head appears to have high susceptibility to elevated IOP. Special instrumentation and measurement techniques are required to document pressure exposure in these eyes. With these techniques, it is possible to obtain an excellent correlation between pressure and the extent of nerve damage [105].

2.2.4.1. Steroid-Induced Ocular Hypertension Ocular hypertension is induced by glucocorticoids, such as dexamethasone, in experimental animal models and a subset of patients treated systemically or topically (steroidinduced glaucoma) [106, 107]. Ocular hypertension in both POAG and steroid-induced glaucoma results from an increased aqueous outflow resistance across the TM, and histopathological findings of the TM in POAG and steroid-induced glaucoma share some similarities [108]. It has been demonstrated that this model shows down-regulation of antioxidant enzymes such as SOD and CAT, causing increased vulnerability to oxidative stress in ocular hypertensive retinas [7]. In addition, Ko et al., [109] demonstrated amplified generation of superoxide anion and increased lipid peroxidation in the retina of a rat ocular

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hypertensive model generated by cauterization of episcleral veins. In another rat model, generated by intraocular hyaluronic acid injection, Moreno et al. [13] also observed increased lipid peroxidation, what was associated with variable retinal SOD activity among studies. Furthermore, retinal SOD response may vary depending on the study protocol [110]. In this model, the results suggest potential vulnerability to oxidative stress in the retina due to a diminution of SOD activity, which is directly involved in cellular protection against oxidative stress of the retinas. However, it remains to be clarified whether these protein alterations are due to the ocular hypertension or to the direct effects of dexamethasone, and future studies should address this important issue. For example, it should be examined whether these alterations take place in other ocular hypertension models.

2.2.4.2. Others Models that Induce Ocular Hypertension The injection of hypertonic saline into episcleral vein [111, 112], cauterization of episcleral veins [113, 114], and laser photocoagulation at TM with or without injection of India ink to enhance laser energy uptake [40] are the currently adopted approaches to produce ocular hypertension models. Except using laser photocoagulation at TM, Wolde- Mussie et al. [115] applied laser treatment at episcleral veins and limbal veins to decrease aqueous outflow and elevate the IOP. However, the understanding of the characteristics of pathological retinal changes in specific models is fundamental for the investigation of glaucoma using those models. Loss of retinal ganglion cells (RGCs) is an important consequence of glaucomatous damage [116]. The ocular hypertension model using cauterization of episcleral veins showed a RGCs loss of approximately 4% per week after IOP elevation [117]. In another model using the injection of hypertonic saline into episcleral veins demonstrated a higher loss of RGC of about 9% per week [118 ]. In a recent study, ocular hypertension induced by unilaterally laser photocoagulation at episcleral veins and limbal veins in adult rats [119 ] show that the loss of RGCs was about 3% per week (25% RGC loss at 8 weeks), which was consistent with previous findings [120] that used the same model. Although the level of IOP elevation in this model was similar to those of injecting saline into episcleral veins, resulted in lower extent of RGC loss. This is probably due to the different experimental approaches adopted.

3. Effects of Antioxidants in Glaucoma There is still much debate about the value of natural antioxidant or vitamin supplementation on ocular diseases such as glaucoma [121, 122].Because the pathogenesis of glaucoma involves various factors, one of which may be oxidative stress, the possibility that natural dietary antioxidants or vitamin supplementations may be beneficial becomes plausible. If diet proves to be beneficial it will be one of the most cost-effective treatments for glaucoma, the incidence of which is expected to increase by 2030, mostly because of the aging population [123]. This disease may in fact be an even more relevant problem in developing countries with social or economical problems. Unfortunately, however, the effect of diet cannot be easily measured due to confounding factors such as timing of diet exposure, level of diet, or intake of other nutrients [124] .

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A number of studies deal with the antioxidant role of polyphenolic compounds in our health. Results are difficult to evaluate and interpret because of the large number of phenolic compounds and the fact that the phenolic content of most foods is not well established. Although there have been major research advancements in the identification and characterization of specific polyphenols [125, 126], many of them remain unidentified. Whereas certain studies reported a significant improvement between green tea consumption and cancer risk [127], others along with a large meta-analysis of epidemiological studies found no such association [128]. This may be a problem of methodology. A close scrutiny of the polyphenolic studies shows that most were in vitro assessments, [129] and only a few are done in vivo. Moreover, the bioavailability or delivery of polyphenols to a specific tissue site is also often not accounted for. Although results of in vitro assessments may be very promising, they do not necessarily reflect in vivo changes. The functional significance of the reports is therefore often not clearly established. Various potential antioxidant compounds are presented in table 11-1. Table 11-1. Antioxidant compounds in glaucoma Compound Tea Wine Ubiquinone (Coenzyme Q 10) Vitamin C Vitamin E PUFA n-3

Mechanism

Effects in Human

Scavenging properties Chelating properties Vasodilating factors Inhibition VEFG Scavenging properties

↓ IOP Wei., et al 1999 ↓IOP,↓neovascularization Garvin et al., 2006 ↓lOP, ↓ lipid peroxidation Nieminen et al., 2005 ↓LDL oxidation, ↓lOP Elmore, 2005 ↓IOP Desmettre, 2005 ↓ IOP Nguyen et al., 2007

↓ NADPH activity ↑ NOS activity ↓ Mitochondrial ROS ↓ TM remodeling ↓ TM cell apoptosis Antiinflammatory

IOP: intraocular pressure; NADPH: nicotinamide adenine dinucleotide phosphate-oxidase; LDL: low density lipoprotein; ROS: Reactive oxygen species; PUFA: polyunsaturated fatty acid; TM trabecular meshwork .

3.1. Polyphenolic Flavonoids 3.1.1. Tea Tea flavonoids have been reported to have powerful antioxidant properties, as a result of their free radical scavenging properties. In fact, the polyphenolic compounds in tea have been shown to act as efficient scavengers for the superoxide anion [130], H2O2 and thereby partially inhibit ultraviolet-induced oxidative DNA damage [131]. Flavonoids present in green tea are able to inhibit the formation of lipid peroxyl radical species and to act as inhibitors of low-density--lipoprotein peroxidation [132]. Myricetin, for example, also functions as a potent and effective neuroprotective agent for photoreceptor cells against oxidative and light damage associated with a diminution of inflammatory and apoptotic biomarkers [133]. Due to their neuroprotective effects [134], the use of green and black tea may prove to be of therapeutic value in the treatment of glaucoma [135].

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Recent in vitro studies on brain membranes showed that epigallocatechin gallate was approximately 10 times more potent than trolox (vitamin E analogue) in attenuating lipid peroxidation caused by the NO donor, sodium nitroprusside. Subsequent immunohistochemical studies revealed that following an intraocular injection of sodium nitroprusside retinal photoreceptors are affected [136]. When epigallocatechin gallate was coinjected, the detrimental effects to the retina caused by sodium nitroprusside were significantly blunted [137]. In agreement with these data, the daily intake of epigallocatechin gallate may help individuals suffering from retinal diseases where oxidative stress is implicated. 3.1.2. Wine Red wines exhibit a stronger antioxidant capacity than white wines due to the higher phenolic content of the first [138]. Polyphenolic flavonoids in wine have been reported to improve endothelial dysfunction and lower the susceptibility of low-density--lipoprotein lipids to oxidation [139]. Impairment in endothelial function may lead to damage to vascular cells and the surrounding tissue. Endothelial dysfunction plays a role in the pathogenesis of a variety of disorders, including glaucoma [140] and cardiovascular diseases [141]. Indeed, red wines strongly inhibit the synthesis of endothelin-1 [142], a vasoactive peptide that plays a crucial role in the pathogenesis of glaucoma. Animal studies have also shown that polyphenolic flavonoids in wine potentially prevent the initiation of atherosclerotic plaque development [143, 144]. Moreover, resveratrol, a polyphenol found in grapes and wine, has been shown to reduce extracellular levels of vascular endothelial growth factor [145].The mechanisms by which polyphenols affect endothelial function is due to their ability to stimulate the production of endothelial NO synthase (eNOS) and promote the production of NO, which induces vasodilation [146]. Red wine polyphenolic flavonoids are available in plant and vegetable sources and have several biological actions, which make these compounds a potentially important agent in the glaucoma therapy. Some problems with respect to bioavailability in clinical trial need to be clarified.

3.2. Antioxidant Vitamins 3.2.1. Thiamin (Vitamin B1) Levels of thiamin have been found to be lower in some glaucoma patients in comparison to controls, and a deficient absorption of this vitamin in these patients has been postulated [147]. Thiamine deficiency has been reported to cause neurodegeneration by inducing various changes in microglia [148], astrocytes [149], endothelial cells, and mast cells [150], leading to neuronal cell death. The brains of thiamine deficient mice show vascular changes, inflammatory responses, and oxidative stress, similar to the brains from patients who die from common neurodegenerative diseases [151]. Recently, the hypothesis that in thiaminedeficient brain [152], vascular factors constitute a critical part of a cascade of events leading to increases in blood-brain barrier permeability to non-neuronal proteins and iron, leading to inflammation and oxidative stress. Inflammatory cells may release deleterious compounds or

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cytokines that exacerbate the oxidative damage to metabolically compromised neurons. Similar mechanisms may operate in the pathophysiology of glaucoma and neurodegenerative diseases in which vascular factors, inflammation and oxidative stress are implicated [153]. 3.2.2. Vitamin C The potential protective effect of vitamin supplementation has not been thoroughly evaluated. Because physiological systems of antioxidant defense are complex it makes sense that optimal functioning requires the availability of numerous antioxidants. Foods provide an even greater array of antioxidants than supplements and a correctly dosed combination of antioxidants, as found naturally, in foods, appears to be more effective [154]. Ascorbate protects low-density lipoprotein cholesterol from oxidative damage and reduces platelet aggregation [155]. By enhancing NO synthase activity, vitamin C is potentially important in lowering blood pressure [156] . The administration of ascorbic acid appears to have a transient hypotonic effect on intraocular pressure in normal eyes. Its effect on glaucomatous eyes is not well defined particularly in the case of the rabbit model [157]. Osmolarity is suggested as a possible hypotensive mechanism and a differential fluid transport rate between the blood and the aqueous humor for the different genotypes is suggested as a possible mechanism for the difference in duration [158]. With respect to doses, the weight of available evidence supports the role of vitamin C in prevention of lens opacities, and the adverse reactions reported to have occurred above the dosage of 1000 mg per day [159]. Vitamin C has been included at 40 mg, equal to the amount of protein, vitamin or mineral that is sufficient for almost every individual. 3.2.3. Vitamin E Mitochondria also produce ROS as a by-product of oxidative phosphorylation. These factors make the mitochondria more susceptible to damage in vitamin E deficiency [160]. Besides serving as an antioxidant, vitamin E has been suggested to be involved in the direct modulation of regulatory proteins, and in the activity of key regulatory enzymes [55]. Apoptosis during hypoxia and oxygen reperfusion can be prevented by vitamin E [57]. There is also some evidence that suggests that vitamin E may reduce apoptosis by means other than antioxidation [161, 162]. In a recent clinical trial, glaucomatous patients were divided into three groups. One group was not supplemented in their therapy. Patients included in the other two groups received 300 and 600 mg/day of oral alpha-tocopherol acetate, respectively. The average differences between the pulsatility indexes and resistivity indexes of both ophthalmic arteries and posterior ciliary arteries of supplemented groups were significantly lower than those not treated at months 6th and 12th. In trial groups, resistivity indexes decreased in posterior ciliary arteries at months 6th and 12th and pulsatility indexes decreased in ophthalmic arteries at the 6th month. In conclusion, alpha-tocopherol deserves attention beyond its antioxidant properties for protecting retina from glaucomatous damage [163, 164]. Recently, some authors described that a mitochondrial complex I defect is associated with the degeneration of TM cells in patients with POAG, and vitamin E and N-acetylcysteine inhibitors can reduce the progression of this condition [165].

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On the other hand, collagen remodeling and apoptosis (associated with an increase in intraocular pressure) are mainly influenced by water-soluble antioxidants such as glutathione [166]. In the case of one matrix collagen type such as elastin, apoptosis and remodeling (correlated with the occurrence of optic atrophy) are particularly influenced by lipid-soluble liposoluble antioxidants such as vitamin E. In addition, the dietary ratio of omega3/omega6 PUFA intake could influence the balance of intraocular pressure. Omega-3 PUFA could influence cyclooxygenase competition through an increase in intraocular pressure reducing synthesis of PG-F2, leading to a decrease in uveo-scleral outflow [167]. The true importance of these factors has not yet been solidly determined and studies are in progress to clarify the real implication of these nutritional factors. 3.2.4. Ubiquinone (Coenzyme Q10) Coenzyme Q10 is a coenzyme for the inner mitochondrial enzyme complexes involved in energy production within the cell. Coenzyme Q10 has been demonstrated to prevent lipid peroxidation and DNA damage induced by oxidative stress [168]. In vivo supplementation with coenzyme Q10 enhances the recovery of human lymphocytes from oxidative DNA damage [169]. These properties are due to ubiquinones free radical scavenging activity [170]. In one study of glaucoma patients, administration of oral ubiquinone was shown to be useful in mitigating cardiovascular side-effects without affecting IOP [171] . Unfortunately, studies examining the effect of ubiquinone on glaucoma are lacking and limit the use of this agent.

4. Conclusions and Perspectives The final common pathway of vision loss in glaucoma is the apoptotic loss of retinal ganglion cells with subsequent degeneration of the optic nerve head. Despite numerous cellular events and pathways have been argued to play major roles in the development of the disease, the exact cause responsible for this alteration is still essentially unknown. The elevated intraocular pressure is not an obligate factor and has been removed from the definition. However, this neuropathy is sensitive to the intraocular pressure; therefore, the medical and surgical treatment is aimed to reduce this parameter, as it halts the progression of the disease in the majority of patients. In primary open angle glaucoma, it is still unknown the exact mechanism that lead to damage of the trabecular meshwork and thereby to an increase in intraocular pressure, nor do we know the exact mechanisms leading to glaucomatous optic neuropathy. Obviously there are a number of factors and mechanisms involved. One of these factors is oxidative stress. At present, studies on the mechanism linking oxidative stress with the development of glaucoma are just arising. Preliminary evidence in glaucoma experimental models indicates that various antioxidant compounds reduce intraocular pressure together with ameliorating the retinal damage. Unfortunately, at this time a pharmacological treatment in humans is not still availability to be recommended for clinical use. Future intensive studies on the effect of these compounds may open up a new therapeutic era in glaucoma. Moreover, the development of randomized controlled double-blind clinical

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trials could help to ascertain the potential therapeutic efficacy of antioxidants in the prevention and treatment of this deleterious disease.

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Index 4 4-hydroxynonenal, 14, 21, 234, 265, 268, 285, 286 4-hydroxynonenal (HNE), 265

A Aβ, 259, 267, 270, 271, 272, 283 AA, 7, 45, 53, 113, 116, 118, 133, 134, 209, 214, 250, 254, 285, 313 abnormalities, 14, 97, 160, 161, 167, 169, 173, 181, 200, 209, 245, 249, 252, 285, 317 absorption, 165, 216, 309 AC, 48, 62, 85, 86, 90, 151, 156, 178, 179, 213, 216, 284, 315 acceptor, 39, 230 accounting, viii, ix, 1, 29, 48, 91, 94, 96, 103, 146, 260, 263 accuracy, 226, 296 ACE, 30, 34, 43, 50, 58, 60, 117, 118, 169, 205, 207, 219 ACE inhibitors, 43, 50, 60, 117, 118, 205, 207 acetate, 310 acetylation, 68 acetylcholine, 13, 19, 26, 29, 34, 51, 53, 70, 85, 120, 190, 204, 263, 264 acquired immunodeficiency syndrome, 117, 129 actin, 73, 116, 128, 313 action potential, 93, 95 activated receptors, 182, 237 activators, 181, 188 active site, 40

acute, vii, 17, 54, 64, 72, 97, 103, 114, 116, 117, 118, 119, 122, 124, 127, 128, 129, 130, 131, 132, 133, 189, 216, 264, 291 acute interstitial nephritis, 119 acute kidney injury, 128 acute renal failure, vii, 17, 122, 124, 127, 128, 129, 130, 131, 132, 133 acute tubular necrosis, 114, 128, 129 acyl transferase, 227 Adams, 89, 90, 219 adaptation, 28 adducts, 82, 142, 178 adenine, 8, 76, 86, 101, 106, 207, 308 adenocarcinoma, 21 adenosine, 36, 76, 244 adenosine triphosphate, 76, 244 adenylyl cyclase, 35 adhesion, ix, 13, 17, 27, 33, 42, 63, 67, 69, 70, 71, 72, 73, 77, 79, 80, 85, 86, 89, 90, 116, 122, 137, 146, 150, 151, 166, 219, 235, 236, 265, 304 adhesive interaction, 146 adipocyte, 162, 163, 169, 178, 179 adipocytokines, 180 adiponectin, 72, 163, 169, 181, 219 adipose, 41, 162, 163, 165, 169, 170, 181, 183, 185, 186, 226, 227, 233, 240, 247 adipose tissue, 162, 163, 165, 169, 170, 181, 183, 185, 186, 226, 227, 233, 240, 247 adiposity, 162, 164, 179, 180 adjustment, 273, 274 administration, 30, 31, 44, 47, 48, 61, 100, 102, 103, 104, 109, 117, 118, 125, 126, 133, 145, 148, 170, 172, 173, 174, 187, 203, 204, 217, 244, 272, 277, 282, 300, 301, 310, 311 adrenal gland, 170

322 adrenal glands, 170 adrenoceptors, 33 adriamycin, 254 adult, 62, 162, 182, 242, 266, 307, 313 adults, 22, 47, 50, 160, 177, 180, 189, 190, 203, 216, 248, 261, 269, 294 advanced glycation end products, xi, 123, 193, 194, 207, 211, 212, 219, 220, 221, 252 advanced glycation end products (AGEs), xi, 193 adventitia, 27, 29, 38, 42, 52, 66, 74, 86 AE, 23, 86, 186, 189, 250, 284 aerobic, 3, 120, 233, 242, 278 aetiology, 286 AF, 57, 89, 92, 93, 94, 95, 96, 97, 98, 99, 102, 103, 104, 106, 107, 219, 317 age, 10, 17, 21, 50, 65, 74, 92, 112, 136, 139, 144, 150, 160, 188, 198, 201, 218, 258, 261, 262, 263, 265, 267, 268, 269, 270, 271, 272, 273, 274, 276, 280, 283, 284, 287, 292, 300, 301, 303, 304, 305, 316, 322 age related macular degeneration, 316 ageing, 112, 210, 258, 261, 284, 296 agent, viii, 2, 4, 6, 7, 11, 18, 26, 32, 33, 40, 44, 79, 100, 118, 125, 183, 205, 206, 207, 232, 239, 241, 244, 256, 272, 308, 309, 311 agents, viii, ix, x, xii, 8, 12, 18, 25, 27, 32, 37, 38, 40, 43, 50, 91, 92, 99, 101, 108, 117, 118, 119, 120, 129, 132, 135, 144, 166, 200, 201, 202, 219, 221, 224, 225, 231, 240, 241, 242, 246, 251, 259, 261, 264, 283, 294, 320 age-related macular degeneration, 303 AGEs, xi, 193, 197, 198, 206 aggregates, 263, 277, 288 aggregation, 71, 73, 100, 200 aggression, 233, 235 aging, vii, xii, 7, 10, 12, 17, 21, 22, 24, 160, 194, 198, 257, 258, 260, 271, 272, 277, 281, 283, 284, 285, 288, 290, 291, 292, 296, 300, 301, 307, 316 aging population, xii, 160, 257, 258, 307 aging process, 17, 22, 24, 258, 260, 272, 281 agonist, 33, 34, 38, 49, 70, 167, 282 aid, 260, 283 AIDS, 117, 118 AJ, 54, 57, 59, 84, 85, 88, 89, 128, 149, 181, 182, 190, 208, 211, 215, 219, 247, 248, 250, 251, 252, 253, 255, 285, 291, 293, 295, 296, 322 AKT, 252 AL, 23, 52, 106, 107, 150, 151, 152, 154, 156, 217, 218, 219, 287, 295, 317 alanine, 132, 137, 174

Index alanine aminotransferase, 174 albumin, 178, 197, 202, 215, 285 albuminuria, 204, 206, 218, 220 alcohol, 22, 49, 166, 173, 224, 231, 232, 245 alcohol abuse, 224 alcohol consumption, 224 alcoholic liver disease, 184, 255 alcoholics, 255 alcohols, 8, 11 aldehydes, 21, 234, 238 aldosterone, 35, 94 allele, 265, 270 alleles, 195 allergic reaction, 119 allograft, 104 allopurinol, 7, 39, 119, 124, 125, 133 allosteric, 34, 54 alpha, 89, 90, 97, 108, 133, 153, 155, 156, 165, 173, 182, 184, 187, 188, 189, 202, 215, 217, 236, 250, 252, 253, 255, 267, 294, 295, 297, 310, 317, 322 alpha-tocopherol, 89, 90, 133, 153, 155, 173, 189, 202, 215, 253, 255, 267, 294, 297, 310, 322 alteplase, 103 alternative, 19, 103, 176, 240, 245, 282, 288, 319 alternative medicine, 319 alters, 40, 184, 189, 295, 304 aluminum, 271 Aluminum, 293 Alzheimer disease, 271, 284, 285, 289, 290, 291, 292, 293, 294, 302 Amadori, 178, 197 amelioration, 18, 104, 175, 240, 253 American Heart Association, 82, 177 American Psychiatric Association, 286 amine, 9, 20, 197, 220, 231, 293 amines, xi, 8, 193 amino, 5, 6, 10, 31, 32, 40, 225, 232, 243, 244, 263, 314 amino acid, 5, 6, 10, 31, 32, 40, 225, 243, 244, 263, 314 aminoglycosides, 123 amniotic, 153 amniotic fluid, 153 amorphous, 263 amygdala, 262 amyloid, 15, 170, 263, 264, 270, 271, 272, 286, 287, 288, 289, 293 Amyloid, 264, 287, 288 amyloid beta, 15, 286, 287, 288, 289 amyloid deposits, 265

Index amyloid fibrils, 272 amyloid plaques, 272 amyloid precursor protein, 287, 288, 289 amyloid β, 263 amyloidosis, 266, 271, 289, 293 AN, 155, 317 anaerobic, 120 analytical techniques, 171 anatomy, 105, 306 Andes, 299 androgen, 189 anemia, 125 angina, 183 angiogenesis, x, 37, 135, 144, 218, 321 angiogenic, 138 angioplasty, 37, 43 Angiotensin, 7, 30, 51, 75, 83, 94, 106, 118, 166, 207, 214, 233, 238, 251 angiotensin converting enzyme, 30, 34, 117, 221 angiotensin II, viii, 13, 23, 25, 26, 45, 51, 52, 53, 55, 57, 58, 69, 75, 93, 94, 98, 113, 152, 206 Angiotensin II, 7, 51, 75, 83, 94, 106, 118, 166, 207, 214, 233, 238, 251 angiotensin receptor blockers, 118 angiotensin-converting enzyme, 48, 54, 221 animal models, viii, xiii, 25, 42, 102, 125, 126, 145, 168, 196, 200, 205, 207, 226, 228, 231, 241, 243, 255, 257, 266, 278, 279, 283, 306 animal studies, 13, 126, 175, 198, 205, 206, 244, 245 animals, 3, 30, 32, 124, 132, 171, 197, 200, 232, 243, 272, 278 Anion, 131 anoxia, 132 ANP, 120 antagonism, 53 antagonist, 31, 40, 185, 206, 267 antagonists, 43, 50, 207, 301 anterograde amnesia, 261 anthracene, 202 anti-angiogenic, 143, 144 antiapoptotic, 217, 319 antibiotics, 117, 123 anticancer, 133 anticancer drug, 133 antidiabetic, 320 antifibrotic, 245 antigen, 195 antigen-presenting cell, 195 antihypertensive agents, 43, 50 antihypertensive drugs, 48, 54, 58, 137

323

anti-inflammatory drugs, 117, 118 Antioxidative, 254, 282, 297 antioxidative activity, 202 antioxidative potential, 87 antiphospholipid antibodies, 136, 143 Antiretroviral, 178 anti-sense, 31 antitumor, 126 anuria, 117 aorta, 30, 32, 33, 47, 54, 62, 83, 93, 145, 200, 204 AP, 85, 96, 107, 130, 133, 179, 186, 211, 235, 250, 289 APC, 195 Apo E, 270 APOE, 292 apoptosis, ix, 10, 20, 28, 29, 33, 42, 73, 77, 91, 92, 96, 102, 107, 109, 114, 116, 126, 145, 146, 148, 156, 157, 165, 182, 201, 210, 219, 229, 232, 234, 235, 236, 241, 245, 249, 250, 251, 255, 265, 278, 279, 280, 285, 286, 287, 303, 306, 308, 310, 311, 315, 321, 322 apoptotic, ix, 91, 109, 140, 171, 207, 225, 237, 238, 303, 308, 311 Apoptotic, 235 apoptotic effect, 303 APP, 259, 261, 263, 264, 266, 267, 270, 271, 272, 287, 289, 293 aqueous humor, xiii, 16, 299, 302, 303, 304, 306, 310, 312, 314, 315, 316 aqueous solution, 19 Arabs, 279 arachidonic acid, 7, 10, 35, 36, 77, 101, 116, 142, 198, 201 ARF, ix, x, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128 arginine, 5, 6, 13, 30, 34, 39, 48, 52, 54, 57, 61, 70, 76, 90, 121, 138, 146, 269, 314 argon, 313, 319 arrest, 28 arrhythmia, ix, 91, 92, 102, 103, 104, 105, 107 arrhythmias, 100, 102, 107, 110 arsenic, 255 arterial hypertension, 32, 57, 61, 92, 166, 173, 288 arteries, x, 16, 30, 32, 36, 43, 47, 53, 58, 64, 66, 72, 82, 135, 137, 139, 142, 146, 148, 154, 204, 214, 218, 310, 320 artery, 13, 19, 32, 37, 41, 53, 54, 55, 58, 69, 70, 85, 87, 92, 96, 99, 103, 108, 110, 115, 131, 139, 143, 144, 145, 146, 148, 154, 157, 173, 183, 189, 190, 194, 203, 214, 216, 270, 292, 321

324 arthritis, 267 ascorbic, 14, 43, 44, 58, 79, 90, 103, 108, 110, 143, 155, 170, 187, 204, 218, 255, 281, 303, 310, 315, 321 ascorbic acid, 14, 43, 44, 58, 79, 90, 103, 108, 110, 143, 155, 170, 187, 204, 218, 255, 281, 303, 310, 315, 321 Ashkenazi Jews, 279 Asia, 273, 275, 294 Asian, 282 aspartate, 137, 167 assessment, 65, 72, 81, 148, 152, 154, 169, 213, 275, 276, 286, 297, 312, 322 astrocytes, 205, 219, 262, 263, 271, 272, 301, 305, 306, 309 Asymmetric dimethylarginine (ADMA), 76, 88 asymptomatic, 12 asynchronous, 94 atherogenesis, ix, 22, 23, 63, 64, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 82, 84, 86, 88, 89, 90, 161, 176, 204, 219, 265, 269, 270 atherosclerosis, vii, viii, 1, 10, 12, 13, 21, 28, 29, 37, 41, 43, 49, 55, 57, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 122, 160, 166, 168, 169, 170, 171, 176, 178, 180, 183, 184, 185, 187, 188, 194, 200, 203, 212, 213, 218, 253, 268, 269, 270, 288, 305, 321 atherosclerotic plaque, 69, 74, 75, 83, 87, 99, 198, 309 atoms, 7 ATP, 76, 95, 116, 120, 228, 230, 306 ATPase, 109, 116, 128 atria, 93, 94, 95, 97 atrial fibrillation, ix, 91, 92, 95, 98, 104, 105, 106, 107, 108, 109, 110 Atrial fibrillation, ix, 91, 92, 105 atrial fibrillation (AF), 104 Atrial fibrillation (AF), 92 atrial natriuretic peptide, 120 atrioventricular node, 93 atrium, 93 atrophy, 269, 305, 311 autoantibodies, 31, 45 autoantigens, 195 autocrine, 51 autoimmune, 119, 161, 195, 196, 209, 217 autoimmune disease, 119 autoimmune diseases, 119 autoimmunity, 196, 315

Index autonomic nervous system, 106 autooxidation, xi, 193, 194 autophagy, 278 autopsy, 276, 280 autoreactive T cells, 195 autosomal dominant, 270, 279 autosomal recessive, 15 availability, 14, 39, 100, 143, 145, 167, 204, 214, 218, 231, 310, 311 awareness, 22 axon, 262, 314 axonal, 258, 305, 306, 319, 322 axons, xiii, 262, 299, 305, 306 azotemia, 112, 113, 115 Aβ, 261, 262, 263, 264, 265, 266, 268, 270, 272

B B vitamins, 291 bacteria, 6, 165 bacterial, 72, 122, 165, 182 bariatric surgery, 179, 180 barrier, 27, 66, 70, 165, 219, 235, 258, 269, 301, 305, 310, 318 basal forebrain, 263 basal lamina, 236 basement membrane, 113, 117, 138 basic research, 187 battery, 149 BBB, 258, 264, 271, 281, 282 BD, 287 beams, 300, 301 beef, 57 behavior, 2, 296 beneficial effect, 33, 43, 46, 49, 80, 92, 125, 133, 145, 170, 173, 174, 175, 202, 205, 208, 271 benefits, x, 58, 104, 136, 171, 174, 197, 240, 244, 270, 274, 282, 283 benign, 225, 246 benzo(a)pyrene, 202 beta cell, 161, 209, 213, 214, 217, 218 beta-blockers, 110 beta-carotene, 90, 155, 202, 215 bile, 231, 242, 245, 255 bile acids, 245 bilirubin, 11 binding, 2, 34, 35, 42, 74, 76, 83, 116, 122, 123, 165, 182, 185, 199, 206, 227, 263, 265, 270, 287, 289 bioactive compounds, 240

Index bioavailability, viii, 13, 25, 26, 29, 32, 35, 36, 37, 41, 42, 43, 44, 48, 50, 57, 70, 74, 76, 77, 121, 138, 184, 200, 308, 309 biochemistry, viii, 1, 21, 191, 210, 285 biogenic amines, 8 biological activity, 4, 19, 79, 252 biological markers, 264, 278 biological processes, 26 biological systems, 1, 9, 18 biomarker, 37, 86, 165, 198, 226, 259 biomarkers, x, xiii, 37, 42, 72, 94, 102, 119, 135, 140, 141, 142, 153, 187, 189, 199, 200, 207, 257, 267, 269, 276, 280, 283, 286, 290, 296, 309 Biometals, 251 biomolecules, viii, 25, 77, 225, 234, 306 biopsies, 97, 150, 199, 234, 242, 243, 244, 245 biopsy, 165, 224, 226, 242, 243, 244, 245 biosynthesis, 218 births, 140 black tea, 309, 320 blastocyst, 150 blepharitis, 316 blindness, xiii, 16, 299, 300 blocks, 7, 13, 58, 132, 206, 210, 220, 229, 267, 281 blood flow, xiii, 28, 66, 75, 115, 120, 121, 139, 140, 141, 142, 150, 152, 299, 305, 317 blood glucose, 58, 199, 207 blood plasma, 83 blood pressure, viii, 25, 26, 27, 28, 30, 33, 34, 37, 39, 40, 42, 44, 45, 46, 47, 48, 49, 52, 56, 58, 59, 60, 61, 62, 65, 109, 136, 169, 171, 172, 176, 190, 237, 310, 317 Blood pressure, 168, 175 blood pressure reduction, 35 blood supply, x, 64, 135 blood urea, 113 blood urea nitrogen, 113 blood vessels, 13, 28, 29, 33, 36, 37, 40, 55, 108, 109, 156, 166, 211, 219, 306, 313 blood-brain barrier, 258, 269, 310, 318 BMI, 269 body fat, 162, 233 body mass index, 136, 143, 144, 162, 164, 224, 242 body weight, 48, 169, 175, 260 Bohr, 289 bonds, 97, 230 borderline, 273 Boston, 128 bovine, 2, 54, 55, 108, 315, 320 bowel, 165

325

bradykinesia, 277 bradykinin, 34, 36, 71, 118 brain, 12, 14, 33, 41, 64, 108, 137, 184, 258, 259, 260, 264, 265, 266, 267, 268, 269, 271, 272, 277, 278, 279, 280, 281, 285, 288, 289, 290, 291, 292, 293, 296, 305, 309, 315, 321 brain damage, 279 brain injury, 291 brainstem, 277 branching, 279 Brazilian, 146 breakdown, 11, 37, 39, 120, 306 breast cancer, 320, 321 bubbles, 34 buffer, 48 bypass, 92, 96, 99, 103, 107, 108, 110, 131 by-products, 228, 234

C Ca2+, 29, 32, 34, 43, 49, 50, 70, 95, 98, 106, 109, 129, 130, 230, 236 calcium, 14, 26, 34, 49, 55, 62, 76, 95, 102, 103, 113, 116, 120, 129, 131, 230, 265, 301 calcium channels, 55 caliber, 28 calmodulin, 34, 54, 76 caloric restriction, 171, 188 calorie, 188, 242 cAMP, 35 Canada, 275 cancer, 12, 17, 18, 59, 60, 61, 117, 189, 190, 231, 268, 308 cancer cells, 320 candidates, 92, 99, 140 cannabinoids, 282 cannabis, 282 capillary, 113, 136, 236, 305 capsule, 113, 137 carbohydrate, 227 carbohydrates, 36, 119, 173, 174, 227, 233 carbon, 4, 7, 44, 79, 118, 231, 232, 253 carbon atoms, 7 carbon tetrachloride, 118 carbonyl groups, 198, 267, 290 carboxyl, 263 carcinogen, 21 carcinogenesis, 7 carcinogenic, 8, 9, 19 carcinogens, 231

326 carcinoma, xii, 9, 223, 224, 252 cardiac function, 109 cardiac myocytes, 33 cardiac output, 118, 136 cardiac surgery, 94, 95, 99, 104, 105, 106, 107, 108, 125, 128, 133 cardiomyocytes, ix, 38, 91, 93, 95, 102, 103, 109, 181 cardiomyopathy, 109, 213, 254 cardiopulmonary, 107 cardiopulmonary bypass, 107 cardiovascular disease, viii, 12, 14, 25, 26, 30, 34, 37, 41, 43, 46, 47, 48, 50, 55, 59, 60, 63, 78, 81, 82, 87, 94, 130, 163, 164, 171, 173, 175, 176, 177, 180, 186, 188, 189, 190, 199, 209, 213, 269, 291, 292, 309, 320 Cardiovascular disease, 12, 64 cardiovascular function, 185 cardiovascular physiology, 36 cardiovascular protection, 99, 171 cardiovascular risk, 13, 30, 43, 46, 55, 57, 65, 72, 81, 167, 172, 173, 174, 176, 177, 186, 269, 270, 283 cardiovascular system, 31, 34, 49, 50, 62, 100 carotene, 59, 78, 79, 80, 153, 273, 275 carotenoids, 48, 79, 90, 143, 153, 154, 273 carotid arteries, 65 carrier, 122, 174, 228 caspase, 229, 230 caspases, 10 CAT, 11, 16, 124, 184, 306 catabolism, 5, 113, 185, 293 catalase, xiii, 11, 41, 42, 92, 124, 132, 139, 142, 150, 172, 188, 196, 203, 217, 232, 234, 265, 272, 299, 303, 316 Catalase, 11, 40 catalysis, 5 catalyst, 4, 84, 123, 217 catalytic activity, 35, 61 catalytic properties, 268 cataract, 303, 321 cataracts, xi, xiii, 193, 198, 299, 300 catechins, 320 catecholamines, 260 cathepsin B, 228, 241 cation, 230 cats, 170, 186 causal relationship, vii, 1 cauterization, 307 CD8+, 72

Index CDK4, 184 CE, 87, 90, 181, 209, 214, 216, 247 cell adhesion, 33, 84, 85, 89, 150, 212, 236 cell culture, 267, 295, 320 cell death, xii, xiii, 16, 20, 96, 103, 122, 129, 145, 148, 181, 203, 224, 225, 228, 235, 246, 250, 265, 277, 281, 285, 299, 301, 302, 303, 309, 319, 320, 322 cell differentiation, 86 cell growth, 26, 28, 30, 37, 55 cell line, 205, 213, 241, 250, 302 cell membranes, 271 cell metabolism, 260 cell signaling, 184 cell surface, 35, 42, 150, 168, 177, 195, 235 cellular adhesion, 71, 236, 304 cellular homeostasis, 10, 229, 303 Cellular response, 316 cellulosic, 122 central nervous system, 32, 33, 53, 137, 260, 306 central obesity, 225 cereals, 49 cerebellum, 266, 268, 290 cerebral blood flow, 258, 264 cerebral cortex, 262 cerebral function, 258, 260 cerebral hypoperfusion, 270 cerebral ischemia, 315 cerebrospinal fluid, 264, 292 cerebrovascular, viii, 63, 64, 264, 269 cerebrovascular disease, viii, 63, 64, 269 ceruloplasmin, 153 channel blocker, 43, 50 channels, 34, 36, 40, 49, 55, 57, 70, 96, 230, 265, 266, 288 chaperones, 230, 277 cheese, 49 chelators, 281 chemical properties, 19 chemical stability, 21 chemiluminescence, 122 chemoattractant, ix, 17, 63, 69, 71, 77, 236, 265 chemokine, 137, 259 chemokines, 71, 73, 97 chemotherapeutic agent, 118 chemotherapy, 118 CHF, 109 childhood, 64, 162 children, 81, 119, 173, 189, 215, 242, 253

Index Chile, 1, 25, 63, 91, 111, 135, 159, 193, 223, 257, 299 Chinese medicine, 240 Chitosan, 177 chloride, 6, 77 chloride anion, 6 CHO cells, 227 cholestasis, 244, 255 cholesterol, 10, 65, 68, 77, 81, 83, 86, 88, 99, 168, 169, 172, 200, 224, 270, 271, 285, 310 cholinergic, 29, 262, 263, 264, 288 cholinergic neurons, 264 cholinesterase, 275 chromatography, 199 chromium, 174, 191, 202 chromosome, 169, 186, 195, 209 chromosomes, 169 chronic disease, viii, 12, 25, 81, 167, 202, 207, 215 chronic diseases, viii, 12, 25, 81, 202 chronic illness, 194 chronic kidney disease, 173 chronic renal failure, 12, 17, 41, 122, 123, 213, 267 cigarette smoke, 211 cigarette smokers, 211 cimetidine, 119 ciprofloxacin, 119 circulation, ix, 20, 91, 92, 94, 104, 118, 122, 138, 140, 143, 151, 156, 165, 226, 317 cirrhosis, xii, 223, 224, 232, 238, 247, 253, 255 cis, 77 cisplatin, 117, 125, 133 Cisplatin, 129 CK, 59, 84, 85, 95, 98, 154, 251, 254, 315, 318, 319 CL, 57, 122, 130, 150, 155, 156, 249, 314 classical, 32, 43, 50, 103, 127, 202, 226, 235, 236, 238, 277, 281 classification, 67, 82, 114, 149, 226 cleavage, 6, 263, 287 clinical approach, 281 clinical diagnosis, 259, 296 clinical presentation, 114, 195 clinical syndrome, 136 clinical trial, vii, viii, ix, x, xii, xiii, 16, 25, 43, 44, 46, 50, 62, 63, 79, 80, 102, 104, 111, 136, 145, 146, 149, 151, 173, 174, 175, 189, 190, 200, 202, 205, 206, 208, 224, 225, 231, 240, 241, 243, 244, 245, 247, 255, 257, 261, 269, 273, 275, 281, 282, 283, 284, 288, 299, 300, 309, 310, 312 clinical trials, vii, viii, ix, x, xii, xiii, 16, 25, 43, 44, 46, 50, 63, 79, 80, 102, 104, 111, 136, 146, 149,

327

151, 173, 174, 175, 190, 200, 205, 206, 208, 224, 225, 240, 241, 243, 244, 245, 247, 257, 261, 269, 273, 281, 282, 283, 284, 288, 299, 300, 312 clinics, 318 clustering, 160 CNS, 260, 264, 265, 276, 306 Co, 53, 128 coagulation, 27, 55, 70, 137 cocaine, 119 Cochrane, 155, 156, 248 coenzyme, 40, 87, 143, 153, 188, 190, 234, 281, 292, 296, 311, 322 co-existence, 93 cofactors, 5, 19, 34, 39, 76, 100, 283 coffee, 319 cognition, 262, 275, 277, 283, 291, 294 cognitive deficit, 262, 263 cognitive deficits, 262 cognitive disorders, 288 cognitive dysfunction, 267 cognitive function, 262, 264, 276, 283, 293, 294 cognitive impairment, 261, 263, 269, 275, 285, 286, 290, 291, 293, 294 cognitive test, 273, 276 cohort, 64, 173, 187, 189, 190, 273, 274, 275, 280, 282, 292, 293 collaboration, 266 collagen, xi, 29, 66, 73, 74, 86, 161, 172, 193, 198, 206, 237, 244, 252, 301, 311 colony-stimulating factor, 69, 84, 86, 236 Columbia, 271, 274 combination therapy, 275 combined effect, 195 communication, 6, 36, 163 communities, 176 community, 202, 215, 258, 271, 286, 292, 293, 294 competition, 311 complement, 107, 122, 202, 241 complement system, 107 complex systems, 27 complexity, 43, 160 compliance, 189, 198 complications, xi, 14, 23, 30, 64, 89, 137, 143, 154, 156, 160, 166, 171, 172, 176, 179, 183, 193, 194, 196, 197, 198, 199, 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 213, 214, 215, 217 components, 2, 3, 7, 9, 23, 27, 28, 46, 50, 52, 64, 68, 72, 73, 77, 100, 104, 141, 160, 164, 168, 169, 170, 172, 174, 177, 202, 237, 238, 277, 286 composition, 28, 164, 271

328 compounds, viii, x, xi, 4, 8, 17, 18, 25, 27, 48, 112, 113, 122, 135, 136, 173, 174, 193, 197, 203, 228, 231, 240, 245, 268, 308, 309, 310, 311, 312 computed tomography, 117 concentration, xi, 2, 3, 4, 10, 16, 26, 31, 32, 44, 48, 94, 95, 96, 102, 103, 112, 115, 116, 121, 124, 125, 137, 142, 143, 150, 159, 161, 165, 196, 203, 206, 209, 214, 230, 261, 302, 303, 305, 314, 315 conception, 139 conduction, ix, 91, 92, 93, 94, 95, 97, 105, 206 conductive, 36 conductivity, 93 confirmatory factor analysis, 177 confounders, 47 congestive heart failure, 13 Congress, iv connective tissue, 28, 29, 66, 67, 237, 238 consciousness, 262 consensus, 239, 267, 284 consumption, ix, xii, 48, 50, 62, 63, 80, 94, 115, 121, 128, 137, 172, 173, 177, 178, 190, 203, 216, 223, 224, 233, 245, 260, 271, 274, 305, 306, 308, 319, 320 contaminants, 122 contractions, 31, 214 control, xi, 12, 14, 19, 22, 23, 27, 28, 30, 34, 49, 51, 53, 55, 57, 70, 79, 81, 103, 106, 118, 142, 144, 161, 169, 170, 171, 176, 183, 193, 195, 198, 199, 200, 202, 204, 206, 240, 272 control group, 103, 142, 144, 204 controlled studies, 149 controlled trials, 81 conversion, 8, 11, 34, 37, 75, 77, 120, 139, 141, 244 convulsion, 136 copper, 2, 18, 21, 151, 203, 251, 271, 275, 294, 304 cornea, 303, 316 corneal epithelium, 303, 315 coronary arteries, 33, 65, 166, 250, 290 coronary artery bypass graft, 92, 96, 103, 108, 110 coronary artery disease, 41, 85, 87, 183, 190, 194, 203, 216, 270, 292 coronary heart disease, 65, 79, 81, 90, 92, 172, 189 correlation, 20, 21, 48, 102, 142, 149, 162, 202, 263, 303, 306 cortex, 259, 264, 266, 268, 272 corticosteroids, 318 cortisol, 187 cosmetics, 322 cost-effective, 307 CP, 22, 107, 248, 254, 255, 284

Index cPLA2, 287 CPR, 6 CR, 108, 129, 131, 150, 181, 185, 218, 251, 289, 315, 318 cranial nerve, 259 CRC, 55 C-reactive protein, 72, 86, 97, 98, 107, 164, 166, 172, 173, 175, 195, 209, 221 creatine, 95, 98, 107 creatine kinase, 95, 98, 107 creatinine, 112, 113, 114, 115, 125, 126, 137, 206 credentials, 160 critically ill, 112, 117, 119, 128 criticism, 267 crossing over, 8 cross-linking, xi, 20, 193, 198, 268 cross-sectional, 181, 196, 209, 216, 273, 274 cross-sectional study, 196 crosstalk, 206 cross-talk, 107 CRP, 72, 195 crystalline, 197 crystalluria, 118 CSF, 263, 264, 267, 268, 269, 280, 285 CT, 24, 248, 322 C-terminal, 287 culture, 313 culture conditions, 313 curcumin, 48, 215, 271, 283, 293 Curcumin, 202, 271 CVD, 64, 65, 68, 70, 78, 80 cycles, 137, 140 cycling, 231 cyclooxygenase, 29, 36, 59, 101, 109, 120, 171, 214, 311 cyclooxygenase-2, 59, 101, 109 cyclophosphamide, 117 cyclosporine, 119 Cyclosporine A, 118 cystathionine, 183 cysteine, 9, 10, 47, 167, 214, 239, 243, 244, 255, 282 cystine, 255 cytochrome, xii, 6, 10, 15, 18, 20, 36, 37, 39, 40, 76, 108, 164, 223, 227, 228, 230, 231, 232, 233, 241, 243, 315, 322 cytokine, 6, 72, 84, 137, 146, 164, 210, 217, 225, 236, 265 cytokines, xi, xii, 17, 29, 32, 67, 72, 73, 75, 96, 97, 120, 164, 193, 203, 223, 234, 236, 237, 265, 301, 310

Index cytoplasm, 40, 71, 225, 277 cytoprotective, 218, 242, 251 cytosine, 9, 20 Cytoskeletal, 312 cytoskeleton, 38, 116, 120, 128, 152 cytosol, 57, 95, 230 cytosolic, 5, 38, 74, 95, 121, 129, 131, 204, 236, 244, 315, 322 cytotoxic, 6, 7, 33, 114, 149, 237 cytotoxicity, 151, 205, 232

D D. melanogaster, 282 daily living, 262, 275 dairy, 49 dairy products, 49 data set, 162 de novo, 7, 32, 226, 227, 232 death, 12, 14, 15, 26, 64, 112, 114, 224, 225, 228, 235, 258, 260, 261, 263, 275, 277, 278, 279, 286, 291, 301, 302, 315 death rate, 258 deaths, 64 decomposition, 6, 217 defects, xiii, 16, 194, 201, 295, 299 defense, vii, ix, 1, 2, 7, 10, 11, 12, 18, 36, 48, 83, 91, 101, 104, 137, 145, 170, 172, 176, 181, 182, 184, 199, 200, 210, 233, 235, 246, 260, 264, 265, 266, 278, 283, 302, 306, 310 defense mechanisms, 18, 137, 145, 199 defenses, 83, 132, 141, 167, 229, 230, 233, 234, 244, 246, 303 deficiency, 29, 34, 39, 56, 79, 115, 119, 122, 133, 160, 171, 202, 231, 254, 255, 277, 292, 303, 309, 310, 321, 322 deficit, 13, 14, 96, 260, 277, 278, 279 deficits, 262, 269, 280 definition, 82, 128, 149, 168, 178, 311 degenerate, 281 degenerative disease, 300 degradation, xi, 35, 41, 75, 83, 122, 124, 193, 206, 241, 250, 253, 267, 272, 279, 282 degradation process, 267 degrading, 42, 316 dehydration, 259 dehydrogenase, 5, 37, 39, 41, 75, 141, 152, 306 delivery, xi, 115, 116, 120, 125, 136, 137, 159, 177, 226, 227, 308 Delphi, 284

329

dementia, 258, 259, 261, 262, 263, 265, 269, 271, 273, 274, 275, 284, 285, 291, 292, 293, 294 demographic factors, 143 demographics, 284 dendrites, 262 dendritic cell, 72 density, 10, 60, 68, 69, 77, 81, 82, 83, 84, 85, 89, 138, 167, 175, 184, 190, 237, 242, 253, 263, 308, 309 deoxyribonucleic acid, 316 deoxyribose, 7 Department of Health and Human Services, 286 depolarization, 10, 93, 228, 266 deposition, 29, 37, 83, 206, 225, 237, 259, 263, 264, 265, 266, 267, 272, 283, 288, 293 deposits, 66, 263, 264, 265, 267 depressed, 171, 186 depression, 201, 203 deprivation, 75, 116, 128, 185 derivatives, xi, 68, 94, 159, 232, 289, 296, 313 desensitization, 109 destruction, 48, 119, 137, 195, 205, 217, 237 detachment, 13, 129 detection, xiii, 17, 33, 72, 143, 144, 216, 245, 251, 257 detoxification, 180, 233 detoxifying, 40, 176 developed countries, xiii, 12, 15, 136, 299 developing countries, 307 dexamethasone, 52, 306 diabetes, vii, xi, 1, 7, 12, 13, 14, 22, 23, 30, 37, 41, 46, 48, 51, 54, 55, 57, 60, 61, 65, 70, 92, 117, 129, 132, 136, 140, 143, 160, 161, 162, 166, 168, 170, 171, 173, 174, 175, 176, 178, 179, 180, 183, 184, 189, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 232, 233, 244, 250, 267, 268, 269, 270 diabetes mellitus, 14, 22, 23, 30, 54, 57, 65, 92, 118, 129, 161, 162, 194, 198, 200, 201, 203, 208, 210, 212, 213, 215, 216, 217, 219, 270 diabetic nephropathy, 123, 132, 202, 204, 211, 214, 220, 221 diabetic patients, xi, 14, 23, 65, 89, 163, 171, 193, 199, 200, 201, 202, 206, 207, 208, 209, 212, 213, 214, 215, 216, 219, 232 diabetic retinopathy, 219, 220 diacylglycerol, 206 Diagnostic and Statistical Manual of Mental Disorders, 286

330 dialysis, 122, 124, 125, 133 diaphragm, 236 diastolic blood pressure, 37, 50 dichotomy, 315 diet, 11, 17, 48, 49, 77, 79, 84, 88, 163, 168, 169, 171, 173, 174, 176, 180, 181, 183, 185, 187, 188, 190, 198, 211, 226, 231, 240, 241, 243, 251, 271, 272, 282, 283, 292, 307 diet composition, 168 dietary, 14, 45, 49, 61, 62, 89, 112, 124, 160, 164, 170, 173, 174, 181, 185, 189, 201, 202, 203, 208, 212, 215, 216, 226, 227, 253, 254, 255, 256, 271, 274, 283, 291, 292, 294, 307, 311 dietary fat, 173, 226, 227 dietary intake, 14, 112, 174, 189, 202, 216, 294 dietary supplementation, 89, 202, 253 diets, viii, 26, 50, 79, 107, 169, 174, 198, 227 differential diagnosis, 114, 260 differentiation, 29, 33, 37, 69, 71, 86, 139, 162, 178, 181, 232, 235 diffusion, 4, 7, 27 dihydroxyphenylalanine, 277 dilated cardiomyopathy, 23 dilation, 26, 29, 42, 55, 93, 94, 219 dimer, 200 dimeric, 76, 243 Dimethylarginine, 269 disability, 282 disease model, 123, 132, 290 disease progression, 195, 265 diseases, vii, viii, xii, xiii, 10, 12, 18, 26, 34, 48, 63, 65, 94, 119, 163, 171, 175, 176, 188, 198, 206, 223, 232, 235, 240, 242, 244, 245, 257, 258, 260, 261, 264, 267, 268, 283, 292, 307, 309, 310, 317 disorder, 14, 15, 24, 92, 93, 104, 106, 160, 226, 261, 276, 277, 283, 300, 314 displacement, 34, 76 distribution, 31, 41, 95, 106, 115, 179, 187, 302 disulfide, 230 disulfide bonds, 230 diuretics, 119 diversity, 185 DNA, xi, xiii, 7, 8, 9, 14, 16, 17, 20, 21, 41, 44, 48, 76, 96, 97, 107, 138, 141, 142, 152, 177, 193, 194, 198, 199, 212, 217, 234, 244, 251, 255, 260, 265, 268, 278, 279, 280, 285, 289, 299, 301, 304, 305, 308, 311, 318, 320, 322 DNA damage, 7, 8, 9, 16, 20, 21, 77, 96, 152, 212, 217, 251, 255, 268, 285, 301, 304, 308, 311, 320, 322

Index DNA repair, 9, 21, 265 DNA strand breaks, 194 dogs, 99, 103, 108, 170, 186, 302, 314 domain structure, 53 donor, 38, 40, 44, 47, 74, 79, 120, 174, 205, 245, 309 donors, 168 dopamine, 266, 272, 277, 278, 296 dopaminergic, 15, 276, 277, 278, 279, 280, 281, 282, 295 dopaminergic neurons, 15, 276, 281, 282 Doppler, 143, 144, 146, 148, 154 dosage, 60, 90, 281, 310 dosing, 145, 148 double bonds, 7, 268 Down syndrome, 265 down-regulation, x, 43, 44, 45, 46, 100, 136, 141, 145, 163, 207, 306 drainage, 304 dream, 106 drinking, 282, 312 Drosophila, 297 drug reactions, 108 drug targets, 183 drug treatment, 103 drug use, 99 drugs, xiii, 16, 65, 92, 99, 100, 103, 117, 118, 119, 122, 129, 155, 166, 176, 199, 201, 205, 219, 231, 240, 243, 299, 300 DSM, 286 DSM-IV, 286 duration, 35, 80, 124, 213, 265, 310 dyslipidemia, xii, 16, 160, 161, 168, 169, 180, 220, 223, 224, 225, 226 dysmetabolic, 180 dysphagia, 284 dysregulated, 60, 120 dysregulation, 17, 163, 225, 250, 305, 317, 322

E EB, 59, 215, 292, 314 eclampsia, x, 15, 24, 135, 136, 137, 139, 149, 150, 153, 154, 156 ECM, 73, 77, 207 edema, 114, 136, 138, 195 Education, 105, 160 eicosanoids, 116 elaboration, xi, 193 elastin, 66, 311

Index elderly, ix, 59, 111, 112, 126, 258, 261, 273, 291, 293, 294, 301 elderly population, ix, 111 elders, 294 electric potential, 95 electrical cardioversion, 103, 108, 109 electrolyte, 114, 117 electrolytes, 112 electron, 4, 5, 6, 7, 10, 35, 37, 38, 39, 40, 44, 46, 74, 76, 79, 96, 100, 129, 130, 152, 174, 196, 198, 200, 207, 228, 230, 232, 236, 264, 281 electron spin resonance, 200 electrons, 2, 5, 39, 41, 48, 74, 75, 76, 228, 229, 230, 231, 281 electrophysiological properties, 93, 97, 103 electrophysiology, 93, 102, 105 elephant, 130, 297 ELISA, 272 EM, 83, 128, 181, 185, 249, 251, 252, 286, 288, 294, 296 emigration, 71 emulsions, 183 encephalopathy, 321 encoding, 227, 270 endocrine, 27, 32, 169, 227 endometriosis, 155 endometrium, 150 endoplasmic reticulum, xii, 49, 168, 184, 223, 228 endothelial cell, xii, 7, 10, 12, 13, 16, 17, 19, 24, 27, 29, 31, 33, 35, 37, 38, 41, 46, 49, 53, 56, 59, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 79, 83, 84, 85, 87, 88, 89, 90, 109, 131, 138, 141, 145, 149, 152, 156, 166, 173, 218, 224, 228, 232, 235, 236, 238, 251, 265, 301, 305, 309, 315, 317, 321 endothelial cells, xii, 7, 10, 12, 13, 17, 19, 27, 29, 31, 33, 35, 37, 38, 46, 49, 53, 56, 59, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 79, 83, 84, 85, 87, 89, 90, 109, 131, 138, 141, 145, 149, 156, 166, 218, 224, 228, 232, 235, 236, 238, 301, 305, 309, 315, 321 endothelial dysfunction, ix, x, 13, 20, 22, 23, 27, 30, 36, 37, 38, 39, 41, 46, 47, 49, 51, 52, 53, 54, 57, 58, 62, 63, 67, 70, 71, 75, 77, 79, 80, 85, 88, 89, 100, 109, 135, 137, 138, 143, 145, 148, 152, 154, 155, 160, 167, 172, 176, 187, 188, 200, 201, 206, 209, 213, 218, 221, 232, 250, 264, 290, 309 endothelial progenitor cells, 13 Endothelin, 31, 52, 62, 156, 320 endothelin-1, viii, 25, 26, 45, 51, 53, 58, 75, 152, 309, 320

331

endothelium, 3, 7, 13, 16, 19, 23, 26, 27, 28, 29, 31, 33, 35, 36, 37, 38, 39, 41, 44, 45, 48, 66, 67, 70, 71, 73, 79, 85, 90, 113, 116, 120, 122, 131, 139, 140, 145, 155, 166, 173, 183, 189, 194, 204, 205, 208, 213, 214, 236, 303, 304, 316, 320, 321 endotoxemia, 165 end-stage renal disease, 61, 114 energy, 10, 15, 116, 160, 161, 162, 164, 179, 185, 225, 226, 227, 228, 230, 271, 284, 305, 306, 307, 311 energy consumption, 305, 306 England, 154 enlargement, 178, 301 enolase, 268 entorhinal cortex, 262, 272 environment, 9, 10, 11, 35, 112, 139, 160, 171, 208, 230 environmental factors, 225, 276 enzymatic, 2, 8, 9, 12, 35, 37, 45, 50, 104, 131, 197, 236, 271 enzymatic activity, 2, 35, 45, 131, 271 enzyme inhibitors, 48, 207 enzymes, viii, 4, 5, 9, 11, 17, 24, 26, 32, 34, 35, 37, 38, 41, 42, 43, 75, 76, 77, 80, 92, 94, 100, 101, 113, 116, 124, 131, 132, 139, 141, 142, 145, 150, 155, 166, 171, 176, 181, 182, 185, 196, 201, 203, 208, 227, 230, 231, 232, 233, 234, 243, 258, 261, 263, 266, 268, 272, 306, 310, 317 eosinophils, 71 epidemic, 160 epidemiologic studies, 81, 271 epidemiology, 81, 105, 128, 283 epidermal growth factor, 178 epigallocatechin gallate, 309, 319, 320 epithelial cell, 113, 114, 116, 130 epithelial cells, 113, 114, 128, 130 epithelium, 16, 116, 303, 315, 321 epitopes, 84, 209 equilibrium, 27, 168 ER, 55, 60, 87, 90, 108, 221, 228, 230, 231, 233, 250, 254, 290, 292 ERK1, 198, 205 erosion, 73 erythrocyte, 2, 121, 125, 200, 216 erythrocytes, 11, 124, 142, 198, 200, 203 erythroid, 125 erythropoietin, 125 ester, 30, 61, 282 esterification, 226 esters, 10, 68, 71, 282

Index

332 estrogen, 171, 282 estrogens, 119 ET, 26, 28, 30, 31, 38, 44, 45, 49, 50, 53, 85, 87, 146 ETA, 31, 45, 53 ethanol, 61, 119, 255, 271 Ethanol, 184 ethnicity, 55 ethylene, 118 ethylene glycol, 118 etiology, 14, 15, 16, 137, 260, 262, 276, 278 EU, 213 Europe, 279 evolution, xii, 68, 184, 213, 223, 226, 238, 272 excision, 265, 289 excitability, 93 excitatory synapses, 301 excitotoxicity, 302 exclusion, 47, 262 excretion, 30, 32, 107, 112, 114, 137, 144 executive function, 261 exercise, 28, 175, 176, 203, 216, 240 exposure, ix, 21, 67, 69, 73, 91, 104, 121, 133, 139, 145, 198, 199, 200, 207, 291, 295, 305, 306, 307 Exposure, 75, 278 extracellular matrix, 26, 28, 37, 66, 67, 73, 94, 97, 123, 237, 301 extraction, 320 extrusion, 18 eyes, 302, 305, 306, 310, 313, 316, 318

F FA, 56, 170, 184, 218, 252, 285, 296, 312 factor analysis, 162, 177 FAD, 5, 6, 34, 74, 76 failure, x, 37, 94, 128, 135, 140, 146, 148, 150, 155, 162, 228, 287 false positive, 144 familial, 15, 68, 177, 186, 270, 277, 278, 287, 296 familial combined hyperlipidemia, 186 familial hypercholesterolemia, 68 family, xi, 2, 5, 34, 36, 40, 60, 70, 77, 159, 171, 215, 230, 241, 248, 279, 288 family history, 70 family physician, 248 Fas, 146, 157 FasL, 146, 157 fasting, 113, 169, 181, 194, 196, 199, 216 Fasting, 152, 210, 252 fasting glucose, 199

fat, viii, xi, xii, 26, 49, 50, 62, 159, 162, 163, 164, 166, 168, 169, 173, 174, 178, 179, 180, 187, 190, 216, 223, 225, 226, 227, 243, 249, 271 fats, 173, 227 fatty acids, xii, 11, 36, 55, 68, 77, 116, 129, 152, 162, 164, 167, 170, 173, 180, 194, 208, 223, 226, 228, 229, 233, 241, 249, 268, 271, 280, 322 Fatty liver, 251 FD, 53, 130, 163 FDA, 125 fear, 259 feedback, 7, 96, 116, 229 feeding, 47, 172 females, 65, 169, 258 ferritin, 126 ferrous ion, 41 fetal, x, 135, 137, 139, 145, 148, 154, 156 fetal growth, 137, 139, 145, 148, 154 fetus, 15, 136 fiber, 237 fibers, 49, 66, 237, 306 fibrillar, 288 fibrillation, ix, 91, 92, 96, 98, 105 fibrinogen, 72 fibrinolysis, 27, 70, 71 fibroblast, 29, 74, 86, 237 fibroblast proliferation, 74 fibroblasts, 5, 29, 31, 38, 66, 69, 74, 77, 87, 237 fibrogenesis, 181, 237, 238, 252 fibronectin, 73, 150, 152 fibrosis, xii, 17, 32, 37, 92, 93, 94, 97, 102, 106, 165, 172, 184, 207, 223, 224, 225, 226, 233, 237, 238, 241, 242, 243, 244, 245, 246, 248, 252, 253, 254 Fibrosis, 237 fibrous cap, 66, 67 fibrous tissue, 237 film, 303 filtration, 113, 115 Finland, 209 fish, 11, 32, 149, 173, 271 FISH, 153 fish oil, 11, 149 fission, 279 FL, 21, 190, 320 flavonoid, 62, 203, 272, 319 flavonoids, 48, 61, 62, 203, 216, 271, 275, 308, 309, 320 flow, xiii, 28, 66, 70, 71, 74, 75, 85, 115, 118, 120, 121, 139, 140, 141, 142, 150, 152, 219, 229, 258, 264, 299, 301, 305, 313, 317

Index fluctuations, 306 fluid, 14, 19, 70, 113, 114, 122, 153, 264, 269, 292, 301, 303, 310, 313 fluid transport, 310 fluorescence, 18, 220 fluoride, 108 focusing, 46, 65, 246 folate, 167, 184, 244, 269, 271, 288, 291 folding, 230 folic acid, 202, 269, 291 food, xi, 159, 162, 174, 185, 198, 211, 231, 292 Ford, 177, 180 forebrain, 262 foreigner, 195 formaldehyde, 220 fortification, 288 Fox, 181, 286 FP, 185, 321 France, 216 free radical, vii, 1, 2, 4, 10, 14, 15, 17, 18, 20, 21, 22, 27, 35, 38, 39, 41, 48, 50, 55, 56, 68, 83, 84, 105, 108, 123, 130, 131, 132, 143, 144, 160, 166, 170, 171, 174, 176, 187, 201, 203, 216, 219, 241, 243, 244, 251, 265, 303, 305, 306, 308, 311, 315, 321, 322 free radical oxidation, 83 free radical scavenger, 219 free radicals, vii, 1, 4, 10, 15, 17, 20, 22, 27, 35, 48, 50, 56, 68, 84, 105, 130, 132, 144, 166, 170, 174, 176, 187, 201, 216, 219, 243, 303, 305, 306 free-radical, 176, 261, 283 fructose, 171, 185, 187, 197 fruits, 49, 50, 124, 173 FS, 152 functional aspects, 251 functional changes, 30, 207 funding, 145 furan, 11 fusion, 279

G G protein, 31, 33 gallbladder, 170 gallbladder disease, 170 gametes, 149 Gamma, 189 gamma-tocopherol, 153, 173 ganglion, xiii, 299, 301, 306, 307, 311, 315, 318, 319, 322

333

gas, 4, 34, 199 gas chromatograph, 199 gastric, 240, 319 gastric ulcer, 319 gastrointestinal, 113 gastrointestinal bleeding, 113 GC, 20, 83, 88, 181, 211, 247 GE, 123, 194, 254, 286, 292, 321 gender, 55, 57, 162, 269 gene, xi, 9, 20, 21, 22, 48, 55, 61, 75, 77, 85, 86, 88, 89, 96, 132, 144, 151, 152, 159, 160, 165, 169, 173, 179, 182, 184, 185, 186, 193, 194, 200, 210, 213, 214, 217, 218, 227, 232, 249, 265, 270, 272, 273, 277, 278, 279, 280, 292, 296, 317, 321 gene expression, xi, 22, 48, 55, 61, 85, 86, 96, 144, 151, 152, 159, 160, 165, 169, 179, 182, 185, 186, 193, 194, 200, 213, 214, 217, 232, 249, 272, 317 gene promoter, 210 gene transfer, 184, 218 generation, vii, ix, xi, xii, xiii, 1, 2, 3, 4, 8, 12, 15, 16, 18, 19, 23, 27, 30, 31, 35, 36, 38, 39, 41, 43, 45, 46, 55, 58, 59, 63, 69, 77, 93, 94, 120, 122, 123, 124, 126, 130, 141, 153, 155, 164, 166, 167, 170, 171, 176, 183, 193, 198, 204, 223, 228, 229, 230, 231, 232, 233, 235, 237, 240, 241, 243, 249, 255, 263, 264, 269, 279, 288, 299, 306, 315 genes, xi, 6, 9, 15, 37, 71, 73, 85, 96, 97, 124, 159, 163, 171, 195, 227, 249, 259, 265, 270, 272, 277, 278, 279, 289, 318, 320 genetic defect, 277, 283 genetic factors, 225, 246 genetic instability, 7 Geneva, 81 genistein, 241, 245, 256 Genistein, 239, 245 genome, 168, 170, 209 genomic, 8, 96 genomic instability, 8 genotoxic, 20 genotype, 270, 274, 292, 303, 316 genotypes, 310 gentamicin, 123, 125, 126, 132, 133 Germany, 312 gestation, 136, 139, 142, 143, 144, 145, 146, 150, 154 gestational age, 144, 150 gestational diabetes, 140 GFAP, 272 GH, 293 GL, 59, 130, 180, 209, 249, 287, 314

334 glaucoma, xiii, 16, 24, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322 glia, 301, 313 glial, xiii, 265, 271, 299, 301, 313, 314 Glial, 301 glial cells, xiii, 265, 299, 301, 313 glial fibrillary acidic protein, 271 glial fibrillary acidic protein (GFAP), 271 gliosis, 277 glomerulonephritis, 17, 24, 206 glomerulus, 17, 113, 118, 119, 120, 122 glucocorticoids, 306 gluconeogenesis, 113, 196, 210 glucose, xi, 44, 47, 58, 61, 75, 79, 113, 123, 159, 161, 163, 168, 169, 170, 173, 174, 176, 179, 185, 187, 193, 194, 196, 197, 199, 200, 202, 207, 208, 209, 210, 211, 213, 214, 216, 227, 233, 238, 240, 244, 248, 251, 270 glucose metabolism, xi, 123, 159, 169, 170, 199, 210, 238, 248, 270 glucose tolerance, 163, 168 glucose-induced insulin secretion, 200 GLUT, 196 glutamate, 265, 266, 285, 289, 300, 301, 302, 313, 314 glutamate receptor antagonists, 301 glutamatergic, 265, 266, 301 glutamic acid, 195 glutamine, 268, 301, 302, 312, 314 glutathione, xiii, 9, 11, 16, 41, 42, 43, 48, 92, 119, 124, 125, 132, 139, 142, 152, 166, 167, 172, 173, 174, 177, 180, 188, 190, 196, 199, 200, 202, 204, 212, 213, 215, 230, 233, 234, 239, 241, 243, 244, 253, 254, 255, 266, 269, 282, 285, 289, 295, 297, 299, 303, 311, 315, 316 glutathione peroxidase, 11, 41, 92, 124, 139, 142, 173, 196, 200, 202, 204, 213, 215, 269, 316 glycation, xi, 15, 187, 193, 194, 197, 198, 208, 210, 211, 212, 220, 221, 233, 238 Glycation, 197, 205, 210 glycemia, xi, 123, 174, 193 glycerol, 126 glycine, 132 glycogen, 173, 196 glycol, 119 glycolysis, 197 glycoprotein, 263 glycoside, 240 glycosides, 319

Index glycosyl, 232 glycosylated, 161, 197 glycosylated hemoglobin, 197 glycosylation, 170, 196, 207, 211, 220 gold, 103 gold standard, 103 government, iv G-protein, 29, 31 gracilis, 55 grading, 249 grafting, 96, 103, 108, 110 granules, 301 granulocyte, 84, 236 grapes, 126, 309 green tea, 187, 272, 282, 283, 297, 308, 320 groups, x, 9, 10, 11, 14, 35, 48, 66, 75, 80, 99, 103, 114, 119, 121, 125, 135, 145, 198, 200, 202, 203, 231, 242, 267, 272, 275, 276, 278, 290, 310 growth, 15, 16, 26, 28, 30, 33, 37, 55, 67, 69, 70, 73, 75, 77, 88, 93, 97, 137, 138, 139, 144, 145, 147, 148, 150, 154, 178, 194, 238, 253, 263, 309 growth factor, 28, 33, 67, 69, 75, 88, 93, 97, 138, 139, 144, 147, 178, 194, 238, 253, 309 growth factors, 28, 33, 67, 75 guanine, 8, 9, 21, 212 guidelines, 65, 81, 280 Guillain-Barré syndrome, 117 gut, 165, 182

H H1, 53 H2, 35, 55 HA, 57, 60, 188, 314, 319 Haj, 131 half-life, 4, 35, 267 halogenated, 231 handling, 230, 260 hands, 259 harm, 179 harmony, 179 Hawaii, 273 HDL, 10, 65, 168, 203 HE, 176, 209, 216 healing, 106 health, xi, 17, 33, 90, 170, 187, 188, 193, 308 Health and Human Services, 286 health effects, xi, 170, 193 hearing, 125 hearing loss, 125

Index heart, ix, 7, 12, 13, 22, 30, 32, 33, 40, 41, 46, 48, 53, 57, 58, 61, 64, 65, 79, 81, 90, 91, 92, 93, 94, 95, 96, 97, 103, 104, 108, 115, 172, 174, 189, 242 Heart, 53, 55, 56, 61, 64, 65, 81, 82, 83, 105, 106, 108, 109, 110, 131, 177, 178, 181, 218, 250, 269, 288, 290, 291 heart attack, 64 heart disease, viii, 61, 63, 64, 65, 79, 81, 93, 172 heart failure, 12, 13, 22, 41, 58, 108, 115, 174 heart rate, 46, 48 heat, 72, 86, 315 heat shock protein, 72, 86, 315 heaths, 305, 306 heating, 9, 198 heavy metal, 118 heavy metals, 118 Helicobacter pylori, 316 hematocrit, 125 hematologic, 119 hematological, 117, 118, 137 hematoma, 66 hematopoietic, 125, 137 heme, 6, 15, 34, 35, 40, 74, 76, 77, 122, 126, 134, 231 heme oxygenase, 15, 126, 134 hemodialysis, 17, 48, 61, 122, 131, 132, 133, 215 hemodynamic, 184, 305 hemodynamics, 53, 118, 128, 149 hemoglobin, 121, 122, 137, 197, 198, 205 hemorrhage, 66, 115 hemostasis, 27, 70 hepatic fibrosis, 238, 252, 253 hepatic injury, 241, 243 hepatic stellate cells, 182, 237, 238, 252 hepatitis, 184, 235, 244, 252 hepatitis a, 235 hepatitis C, 184, 252 hepatocellular, xii, 223, 224, 225, 229, 252 hepatocellular carcinoma, xii, 223, 224, 252 hepatocyte, xii, 164, 224, 225, 226, 228, 229, 230, 233, 234, 235, 236, 238, 241, 242 hepatocytes, xii, 8, 11, 162, 164, 165, 223, 226, 228, 232, 233, 236, 237, 245, 272 hepatoma, 250 hepatorenal syndrome, 115 hepatotoxic drugs, 224 hepatotoxicity, 251, 252, 253, 255 herbal, 320 heredity, 55, 177 heterogeneity, 50, 80

335

heterogeneous, 69, 115, 208, 258 high blood pressure, 13, 27, 38, 49, 136, 172 high fat, 231, 241 high pressure, 3 high resolution, 54 high risk, 14, 126, 145, 149, 156, 161, 173 high-density lipoprotein, 10, 184 high-fat, 163, 168, 169, 180, 190, 226, 227 high-performance liquid chromatography, 199 high-risk, 144, 145, 146, 176 hip, 162 hippocampal, 261, 269, 287, 291 hippocampus, 262, 264, 266, 268, 272, 288, 289, 290 histamine, 34, 35 histological, 82, 126, 140, 180, 238, 240, 241, 244, 249, 254, 262, 263, 265, 276, 302 histology, 174, 226, 242, 253 histopathology, 290 HIV, 177, 178 HK, 149, 212, 288 HLA, 195 Holland, 51, 56, 87, 88 homeostasis, viii, 12, 26, 27, 29, 41, 63, 64, 70, 74, 95, 113, 114, 117, 161, 163, 179, 187, 265, 279, 286, 295, 315 homocysteine, 51, 152, 166, 167, 171, 174, 184, 245, 290, 291 Homocysteine, 167, 183, 184, 288, 291 Honda, 180, 284 honey, 49 hormone, 113, 171, 179, 185, 188, 272 hormones, 26, 27, 29, 33, 113, 225 hospital, ix, 91, 92, 128, 146 hospital stays, ix, 91 hospitalization, 112 hospitalized, ix, 111, 112, 122, 126 host, 7, 83 HR, 54, 87, 133, 185, 316 HSP60, 72 human condition, 167 human immunodeficiency virus, 162 human neutrophils, 202 humans, viii, xi, 5, 9, 25, 32, 33, 36, 39, 43, 44, 48, 51, 55, 58, 69, 76, 79, 89, 125, 126, 163, 164, 168, 170, 174, 175, 189, 193, 198, 200, 207, 226, 231, 244, 246, 273, 278, 304, 306, 311, 321 hyaline, 224, 242, 252 hybrids, 169 hydatid, 150 hydatid mole, 150

336 hydro, 11, 112, 190, 231 hydrocarbons, 231 hydrogen, viii, xiii, 5, 7, 10, 23, 25, 26, 28, 35, 39, 41, 46, 48, 51, 55, 56, 77, 117, 120, 163, 194, 212, 228, 229, 231, 278, 299, 315, 316, 320 hydrogen peroxide, viii, xiii, 5, 23, 25, 26, 28, 35, 39, 41, 46, 48, 51, 55, 56, 77, 120, 163, 194, 212, 228, 229, 278, 299, 315, 316, 320 hydrolases, 231 hydrolysis, 33 hydrolyzed, 301 hydroperoxides, 11, 199 hydrophilic, 11, 190 hydrophobic, 4, 11, 166, 242 hydrostatic pressure, 115, 116, 318 hydroxide, 4 hydroxyl, 4, 9, 37, 41, 48, 108, 120, 126, 194, 205, 228, 278, 281, 288, 306, 319 hydroxylation, 39, 75, 234, 244, 268 hyperactivity, 170 hypercholesterolemia, 13, 57, 70, 79, 86, 88, 89, 90, 168, 254, 270 hyperglycaemia, 180 hyperglycemia, 123, 160, 161, 170, 172, 174, 179, 189, 194, 196, 197, 198, 199, 200, 203, 207, 208, 210, 211, 212, 214, 225, 227 hyperhomocysteinemia, 138, 168, 183, 290 hyperinsulinemia, 161, 168, 170, 172, 174, 178, 194, 227 hyperkalemia, 114, 117, 124 hyperlipidemia, 17, 67, 163, 172, 194, 206, 213, 243, 246, 269 Hypertension, v, 12, 22, 23, 25, 26, 27, 42, 43, 49, 51, 52, 53, 55, 57, 58, 59, 60, 61, 62, 86, 88, 109, 136, 151, 153, 155, 166, 178, 187, 189, 214, 219, 306, 307 hypertensive, viii, 22, 23, 25, 26, 30, 37, 39, 40, 42, 44, 46, 47, 48, 50, 54, 56, 57, 58, 60, 61, 62, 74, 102, 108, 109, 145, 149, 153, 154, 155, 156, 169, 170, 182, 186, 188, 190, 214, 218, 306, 315 hypertonic saline, 306, 307 hypertriglyceridemia, 162, 169, 186, 200 hypertrophy, 30, 32, 39, 52, 73, 94, 97, 102, 106, 206 hypocholesterolemic, 253 hyponatremia, 114, 117 hypoperfusion, x, 115, 135, 137, 139, 146, 264 hypotension, 31 hypotensive, 30, 48, 172, 310

Index hypothesis, 9, 17, 23, 24, 38, 40, 45, 47, 55, 67, 68, 70, 78, 82, 84, 94, 98, 99, 140, 143, 144, 148, 160, 162, 168, 175, 176, 189, 196, 201, 203, 225, 245, 251, 267, 268, 270, 272, 276, 283, 285, 287, 288, 301, 302, 305, 309 hypovolemia, 118 hypoxemia, 260 hypoxia, x, 32, 75, 96, 102, 103, 109, 115, 116, 121, 129, 131, 135, 138, 140, 141, 303, 310 Hypoxia, 129, 139, 317 hypoxic, 106, 117, 139

I iatrogenic, 136 IB, 57, 60, 154, 314 ICAM, 71, 73, 85, 145, 156, 179, 236 ice, 272 ICU, 128 id, 146 identification, 33, 140, 160, 283, 290, 308 identity, 3 idiopathic, 23, 277 IFN, 73, 236 IGF, 150 IGF-I, 150 IgG, 199 IL-1, 72, 79 IL-6, 44, 72, 98, 145, 196 IL-8, 73 imaging, 70, 291 imbalances, 211, 225 immune activation, 72, 240 immune cells, 234, 236, 237, 238 immune response, 33, 166, 195, 232, 235 immune system, 2, 86, 225, 233, 234 immunity, 145, 210 immunoglobulin, 117 immunohistochemical, 150, 309, 321 immunological, 67, 233 immunomodulation, 236 immunomodulatory, 242 immunosuppressive, 118, 195 immunosuppressive drugs, 195 impaired glucose tolerance, 168 in situ, 38, 320 in utero, 139 in vitro, 8, 9, 15, 41, 46, 48, 53, 69, 73, 78, 79, 83, 89, 102, 121, 123, 124, 126, 130, 132, 148, 156,

Index 195, 198, 203, 207, 216, 219, 229, 232, 244, 245, 279, 282, 304, 308, 309, 316, 320 in vivo, vii, 1, 2, 9, 13, 36, 37, 38, 44, 48, 52, 58, 69, 77, 78, 79, 83, 88, 89, 116, 126, 163, 171, 184, 195, 197, 198, 201, 219, 220, 221, 229, 232, 238, 241, 242, 253, 267, 279, 290, 304, 308, 320, 321 inactivation, 9, 11, 13, 37, 38, 61, 119, 146, 205, 207, 208, 230, 234 incidence, 12, 15, 45, 59, 64, 99, 103, 104, 105, 112, 118, 133, 136, 145, 149, 173, 202, 215, 274, 276, 307 inclusion, 202 income, 273 India, 153, 307 Indian, 271 Indians, 162, 191 indices, 213 indigenous, 202 indirect effect, 29 inducer, 172 induction, 16, 26, 48, 73, 75, 77, 80, 85, 124, 126, 148, 155, 172, 184, 197, 198, 204, 211, 212, 231, 232, 233, 236, 238, 260, 269, 319 industrial, 231 infants, 150 infarction, 141, 305 infection, 48, 119, 235 infectious, 119 Infiltration, 236 inflammation, ix, 2, 3, 9, 18, 19, 33, 37, 42, 55, 61, 66, 70, 71, 72, 86, 91, 92, 94, 96, 97, 99, 104, 105, 109, 137, 145, 156, 160, 164, 165, 168, 171, 172, 174, 181, 187, 188, 189, 202, 209, 212, 213, 216, 224, 226, 234, 235, 236, 237, 241, 242, 243, 245, 246, 251, 252, 259, 267, 279, 302, 310, 321 inflammatory, ix, xii, 3, 17, 26, 27, 29, 33, 55, 63, 67, 71, 72, 73, 74, 75, 78, 80, 82, 85, 93, 97, 99, 106, 107, 112, 114, 116, 117, 118, 120, 130, 140, 151, 154, 160, 164, 165, 166, 168, 170, 174, 176, 179, 183, 185, 194, 198, 203, 205, 207, 209, 219, 221, 223, 225, 227, 233, 234, 235, 236, 237, 238, 239, 240, 245, 252, 259, 265, 267, 269, 271, 288, 308, 309 inflammatory bowel disease, 267 inflammatory cells, 74, 116, 198, 237 inflammatory disease, 71, 82, 240 inflammatory mediators, ix, xii, 63, 223, 234, 235, 236, 238, 252

337

inflammatory response, xii, 67, 72, 73, 75, 78, 80, 120, 140, 151, 154, 170, 179, 224, 235, 236, 238, 309 inflammatory responses, 309 ingest, 79, 227 ingestion, 24, 203 inherited, 185, 258, 278, 279 inhibition, xi, 9, 30, 34, 36, 47, 49, 51, 53, 60, 69, 70, 71, 76, 77, 79, 87, 89, 99, 102, 118, 121, 124, 132, 139, 159, 199, 205, 206, 219, 220, 221, 228, 232, 240, 246, 279, 281, 295, 320, 321 inhibitor, 7, 31, 34, 39, 40, 54, 57, 58, 72, 76, 84, 87, 88, 118, 124, 126, 139, 150, 169, 202, 216, 218, 219, 220, 221, 231, 269, 275, 281, 292, 296, 314 inhibitors, 17, 29, 31, 48, 75, 108, 117, 118, 162, 203, 204, 205, 207, 212, 219, 221, 232, 295, 308, 311 inhibitory, 97, 232, 266 inhibitory effect, 232 initial state, 225 initiation, 9, 64, 68, 86, 95, 204, 302, 309 injection, 126, 203, 306, 307, 309 injections, 279, 314 injuries, 17, 94, 102, 112, 211 injury, ix, x, 2, 13, 15, 17, 20, 22, 23, 26, 28, 29, 36, 47, 55, 58, 60, 67, 70, 73, 82, 91, 93, 94, 96, 104, 106, 107, 108, 111, 113, 114, 115, 116, 119, 120, 121, 122, 123, 124, 125, 126, 127, 129, 130, 131, 132, 134, 151, 164, 165, 182, 184, 188, 194, 197, 204, 206, 207, 208, 211, 225, 229, 234, 236, 237, 241, 243, 246, 249, 251, 252, 253, 260, 261, 264, 279, 282, 301, 302, 305, 306, 313, 315, 317, 318 innate immunity, 71 innervation, 264 inorganic, 4 iNOS, 6, 33, 76, 88, 101, 138, 141, 204, 218, 232, 235, 236, 269, 279 inositol, 32 INS, 205 insertion, 6, 9 insight, 26, 280 instability, 7, 124, 305 institutionalization, 275 instruments, 267 insulin, xi, xii, 14, 23, 32, 61, 75, 159, 160, 161, 162, 163, 164, 166, 168, 169, 170, 172, 174, 175, 176, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189, 190, 193, 194, 195, 196, 197, 200, 202, 203, 204, 208, 210, 211, 213, 214, 215, 216, 217, 219,

338 223, 224, 225, 227, 233, 237, 238, 239, 240, 246, 249, 250, 251, 270 insulin resistance, xi, xii, 61, 159, 160, 161, 162, 163, 164, 166, 168, 169, 170, 172, 175, 178, 179, 180, 182, 185, 186, 188, 189, 190, 196, 202, 203, 208, 210, 215, 216, 223, 224, 225, 227, 233, 237, 238, 239, 240, 246, 249, 250, 251, 270 insulin sensitivity, 162, 170, 176, 179, 187, 219 insulin signaling, 186 insulin-producing cells, 194, 210 insults, 114, 236 integrins, 71, 116, 129, 150 integrity, viii, 11, 13, 25, 27, 28, 88, 114, 116, 120, 128, 165, 190, 208, 230, 304 interaction, xi, 18, 26, 27, 28, 36, 67, 108, 121, 142, 146, 148, 159, 167, 190, 193, 198, 208, 264, 266, 316 interactions, 22, 23, 27, 32, 54, 55, 70, 85, 146, 157, 160, 181, 182, 281, 316, 317 intercellular adhesion molecule, 71 interference, 168, 238 interferon, 72, 195, 203, 236 interferon-γ, 72, 195, 203 interleukin, 44, 58, 73, 97, 98, 156, 196, 203, 219, 235, 236, 271 interleukin-1, 203, 219, 235, 236, 271 interleukin-6, 44, 58, 73, 97, 98, 156, 196, 236 intermolecular, 198 interstitial, 27, 116, 119, 130 interstitial nephritis, 119, 130 interval, 282 intervention, 46, 50, 143, 166, 167, 173, 194, 196, 200, 240, 241, 242, 243, 244, 245, 255, 267, 271, 283 intestine, 3 intima, 66, 67, 68, 71, 72, 77, 82, 84 intoxication, 108, 117 intracellular signaling, xi, 17, 126, 193 intramuscular, 126 intramuscular injection, 126 intraocular, xiii, 16, 299, 300, 301, 303, 304, 307, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 319, 322 intraocular pressure, xiii, 16, 299, 300, 301, 303, 304, 308, 310, 311, 312, 313, 314, 315, 317, 318, 319, 322 intrauterine growth retardation, 150 intravascular, 137 intravenous, 44, 59, 125, 133, 282, 297 intravenously, 48

Index intrinsic, 36, 92, 93, 102, 114, 122, 131, 230, 234 invasive, 80, 139, 150 invertebrates, 174 investment, 81 ion channels, 96 ions, 8, 19, 259 IOP, 300, 301, 304, 305, 306, 307, 308, 311 IP, 156, 157 IR, 186 Iran, 243 iris, 303, 316 iron, xii, 4, 34, 76, 123, 125, 133, 151, 180, 223, 251, 278, 281, 296, 304, 310, 320 iron deficiency, 133 IS, 60, 129, 216 ischemia, ix, x, 3, 5, 7, 19, 20, 22, 91, 92, 94, 95, 97, 102, 103, 104, 109, 111, 115, 116, 117, 120, 121, 124, 126, 128, 130, 131, 132, 134, 137, 141, 150, 151, 181, 188, 204, 218, 253, 305, 306, 314, 315 ischemia reperfusion injury, 134 ischemic, viii, 37, 43, 63, 64, 94, 109, 114, 115, 116, 117, 120, 121, 124, 127, 129, 130, 131, 132, 305 ischemic heart disease, viii, 63, 64 ischemic stroke, 37, 43 Islam, 133, 210 isoenzymes, 6 isoflavones, 202, 245 isoforms, 5, 34, 38, 40, 56, 76, 123, 214, 231, 270, 287 isolation, 176 isomers, 171 Italy, 224, 248, 300, 312 IV collagenase, 156

J JAMA, 59, 60, 81, 90, 108, 128, 177, 286, 293, 294, 319 Japan, 224 Japanese, 212, 248, 254, 273 JI, 177 Jordan, 61 JT, 18, 56, 87, 150, 182, 217, 220, 254, 285, 287, 292, 294, 295, 316 Jung, 52

K K+, 34, 36, 49, 70, 116

Index kainic acid, 289 kappa, 31, 138, 170, 187, 232 kappa B, 31, 138, 170, 187, 232 ketones, 164 KH, 52, 108, 154, 155, 157, 211, 255, 288, 291, 293, 294 kidney, 7, 17, 32, 33, 54, 61, 62, 112, 113, 114, 115, 116, 118, 119, 120, 121, 123, 124, 126, 128, 129, 130, 131, 132, 137, 173, 184, 187, 198, 204, 220 kidney stones, 17 kidneys, 115, 130, 134, 148 kinase, 6, 15, 34, 70, 89, 95, 123, 154, 179, 184, 194, 206, 214, 232, 278, 279 kinase activity, 107, 278, 279 kinases, 37, 53, 230 kinetics, 198 King, 154, 155, 209, 314, 319, 322 kinins, 34 KL, 22, 83, 130, 156, 179, 185, 189, 249 knockout, 30, 33, 168, 200, 206, 267, 280, 289 Korean, 182 Krebs cycle, 76

L LA, 53, 59, 61, 107, 153, 169, 177, 186, 190, 217, 248, 249, 250, 251, 286, 288, 291, 314, 315, 318 labor, 155 lamellae, 300 lamina, 26, 66, 71, 75, 87, 139, 236, 313 laminar, 26, 71, 75, 87, 313 Laminar, 75, 85 laminin, 150 language, 261 laser, 306, 307, 313, 319 latency, 300 late-onset, 270, 292 late-onset AD, 270 late-stage, 262, 285 LC, 52, 55, 129, 154, 155, 190, 255, 297, 319 L-carnitine, 282 LDL, ix, 10, 21, 46, 63, 65, 67, 68, 69, 72, 73, 75, 77, 78, 79, 82, 84, 89, 125, 133, 138, 163, 166, 167, 168, 172, 173, 175, 183, 198, 242, 308 leakage, 46, 315 left ventricle, 34 lens, xi, 193, 197, 198, 303, 310, 321 lenses, 211 leptin, 139, 150, 163, 168, 169, 181, 200, 252

339

lesions, 66, 67, 68, 69, 72, 73, 74, 75, 76, 77, 82, 83, 84, 87, 88, 126, 166, 206, 249, 319 leukocyte, 27, 33, 67, 70, 317 Leukocyte, 151 leukocytes, 18, 42, 58, 71, 73, 97, 120, 138, 140, 141, 237, 318 leukotrienes, 129 levodopa, 277, 282 Lewy bodies, 276, 277, 279 LH, 106, 107, 169, 284, 292, 321 liberation, 260, 279 life expectancy, 258, 283 life forms, 3 lifespan, 188, 261 lifestyle, ix, 12, 57, 111, 253 lifestyle changes, 12 lifetime, 10, 258 ligand, 35, 71, 157, 188, 315 ligands, 71, 206, 208 likelihood, 143, 149, 196, 275 limitation, 235 limitations, 168, 259, 284 linear, 164, 173 linkage, 138, 169, 207 links, 7, 166 linoleic acid, 10 lipase, 185 lipemia, 169 Lipid, 9, 10, 21, 58, 81, 86, 88, 89, 90, 152, 153, 164, 181, 198, 208, 213, 247, 250, 268, 312 lipid metabolism, 170, 173, 183, 224, 225, 227, 230, 287 lipid oxidation, 41, 77, 171, 198 lipid peroxidation, x, xi, 4, 7, 9, 11, 14, 21, 22, 24, 41, 42, 46, 57, 79, 83, 90, 99, 108, 119, 123, 124, 126, 135, 138, 140, 141, 142, 143, 144, 151, 153, 155, 159, 164, 166, 171, 172, 174, 181, 187, 188, 198, 200, 203, 212, 213, 225, 226, 228, 231, 234, 241, 245, 251, 254, 261, 265, 266, 267, 268, 272, 278, 279, 280, 286, 289, 290, 300, 305, 306, 308, 309, 311, 321 lipid peroxides, 153, 156, 164, 173 lipid profile, 213 lipids, xi, xii, 4, 17, 41, 44, 48, 59, 67, 68, 73, 75, 76, 83, 116, 119, 142, 163, 171, 174, 189, 193, 197, 198, 202, 223, 225, 226, 227, 232, 236, 260, 267, 271, 277, 291, 303, 306, 309 lipodystrophy, 162, 178 lipolysis, 170, 186, 236 lipooxygenase, 69, 77

340 lipopolysaccharide, 236, 250 lipoprotein, 10, 67, 81, 82, 83, 84, 85, 86, 87, 89, 90, 161, 167, 175, 184, 189, 212, 230, 253, 308, 310 Lipoprotein, 83 lipoproteins, 10, 68, 69, 77, 82, 83, 84, 100, 138, 154, 163, 183, 184, 190, 203, 216, 224, 242, 249 liposomes, 90, 181 lipoxygenase, 32, 36, 38, 82, 84, 88, 89, 120 liquid chromatography, 190, 199 lithium, 117 liver, x, xi, xii, 2, 7, 18, 44, 79, 108, 118, 131, 135, 137, 150, 159, 161, 162, 163, 164, 165, 168, 170, 172, 174, 177, 178, 179, 180, 181, 182, 184, 185, 188, 190, 207, 210, 223, 224, 225, 226, 227, 228, 229, 232, 233, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 272 liver cells, 225, 228 liver cirrhosis, 255 liver damage, 226, 227, 253 liver disease, xii, 118, 180, 181, 182, 184, 185, 190, 223, 224, 225, 229, 232, 237, 238, 239, 241, 243, 244, 247, 248, 249, 250, 251, 252, 253, 255 liver enzymes, 150, 182, 243 liver failure, 224 liver transplant, xii, 223 liver transplantation, xii, 223 LM, 22, 51, 57, 82, 86, 90, 109, 130, 149, 150, 180, 249, 250, 285, 287, 296, 318, 321 localization, 42, 56, 72, 150, 152, 188 location, 38, 93, 100, 106, 108, 265 locomotor activity, 297 locus, 195 London, 249, 251 longitudinal study, 154, 273 losses, 100 lovastatin, 119 low fat diet, 226 low risk, 145 low-density, 10, 82, 84, 85, 86, 87, 89, 90, 125, 184, 188, 189, 212, 226, 308, 309, 310 low-density lipoprotein, 10, 82, 84, 85, 86, 87, 89, 90, 125, 184, 188, 189, 212, 226, 310 low-density lipoprotein receptor, 188 LPS, 165 lumen, 64, 66, 67, 113, 116, 182, 230, 236, 301 luminal, 28, 31, 66, 70, 113, 165 lung, 9, 20, 21, 108, 132, 211, 320 lung cancer, 211, 320 lungs, 33

Index lupus, 119 lutein, 79 LV, 86, 218 lycopene, 79, 90, 203 lymph, 195 lymph node, 195 lymphatic, 169 lymphocyte, 195, 254 lymphocytes, 67, 71, 272, 286, 305, 311, 317 lysine, 9, 198, 215

M M1, 316 machinery, 227, 276, 279 macromolecules, 260, 268, 280 macronutrients, 292 macrophage, 20, 33, 68, 69, 71, 82, 83, 84, 166, 184, 253 macrophages, 8, 29, 33, 37, 64, 66, 67, 68, 69, 70, 71, 72, 74, 76, 77, 84, 85, 87, 141, 166, 183, 198, 203, 216, 234, 236, 237, 251, 252 macular degeneration, 303, 316 magnesium, 137, 202 magnetic, 210, 291 magnetic resonance, 18, 291 magnetic resonance imaging, 291 Maillard reaction, 197, 198, 211 maintenance, 10, 20, 27, 29, 34, 41, 48, 86, 112, 114, 115, 116, 122, 123, 128, 161, 229, 235, 258, 279 Maintenance, 230 maladaptive, 97 males, 169, 258 malignant, 234 malnutrition, 259 malondialdehyde, 9, 15, 21, 152, 200, 201, 204, 234, 265, 302, 316 malondialdehyde (MDA), 9, 265 Mammalian, 132 mammalian cells, 2, 4, 11, 61, 233 mammals, 6, 18, 41, 250 management, viii, xiii, 16, 25, 43, 49, 81, 128, 129, 136, 137, 150, 176, 177, 199, 209, 224, 230, 243, 247, 286, 299, 300 manganese, 40, 132, 232, 250 manganese superoxide, 132, 250 manganese superoxide dismutase, 132, 250 manipulation, 38, 164, 200 mannitol, 117, 129 MAO, 281, 296

Index MAPK, 33, 109 mapping, 186 mass spectrometry, 54, 190, 199 mast cell, 309, 321 mast cells, 309 maternal, x, 15, 16, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 148, 149, 150, 151, 153, 154, 155, 156 maternal age, 136 matrix, 26, 28, 37, 40, 66, 67, 72, 73, 94, 97, 123, 147, 237, 280, 300, 301, 311, 313, 317 matrix protein, 123 maturation, 171, 264, 266 MB, 109, 189, 190, 284, 294, 296, 314, 315, 317, 320 MCI, 261, 267, 268, 269, 275 MCP, 69, 71, 73 MCP-1, 69, 71, 73 MDA, 9, 14, 142, 144, 245, 267, 268 meals, 50, 161, 162 mean arterial blood pressure, 48 measurement, 19, 20, 85, 302, 306 measures, 118, 194, 202, 203 meat, 50 mechanical stress, 28, 305 media, 13, 42, 66, 72, 117, 139 median, 276 mediation, 183, 234 mediators, viii, ix, xii, 22, 25, 26, 28, 29, 36, 41, 50, 55, 63, 93, 96, 97, 103, 104, 122, 126, 127, 143, 164, 203, 210, 223, 225, 231, 234, 235, 236, 237, 238, 249, 252, 283 medications, 61, 113 medicinal plants, 240 medicine, 99, 202, 240, 319 Mediterranean, 17, 49, 62, 173, 292 melanin, 316 melatonin, 62, 272, 319 membrane permeability, 117 membranes, 4, 5, 11, 19, 38, 41, 100, 104, 108, 116, 125, 129, 166, 171, 230, 231, 232, 241, 259, 264, 265, 281, 289, 301, 309 memory, 71, 216, 261, 262, 272, 273, 284, 293 memory deficits, 216, 293 memory performance, 273 men, 44, 59, 60, 160, 180, 182, 187, 202, 215, 216, 258, 273, 275, 293 menopause, 171 mercury, 118 mesangial cells, 123

341

mesenteric vessels, 200 messenger RNA, 82 messengers, ix, 26, 37, 91, 92 meta-analysis, 46, 90, 99, 105, 146, 173, 177, 189, 190, 292, 308, 320 metabolic, xi, xii, 1, 10, 12, 14, 17, 24, 38, 75, 115, 126, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 184, 185, 186, 187, 188, 189, 190, 194, 202, 208, 223, 224, 225, 226, 227, 233, 238, 240, 244, 246, 247, 248, 250, 260, 300, 314 metabolic disorder, 163, 194, 224 metabolic disturbances, 194 metabolic dysfunction, 174 metabolic pathways, 14, 161, 166, 176, 208 metabolic rate, 17, 260 metabolic syndrome, xi, xii, 14, 24, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 182, 184, 185, 186, 187, 188, 189, 190, 223, 224, 225, 238, 247, 248 metabolism, vii, ix, xi, 2, 4, 7, 10, 15, 24, 36, 39, 75, 83, 95, 111, 112, 116, 119, 120, 123, 128, 159, 160, 164, 165, 166, 167, 169, 170, 173, 174, 176, 183, 184, 186, 187, 193, 194, 198, 199, 205, 210, 224, 225, 226, 227, 228, 230, 233, 234, 238, 243, 248, 260, 261, 270, 271, 279, 280, 284, 285, 287, 295, 300, 315, 321 metabolite, 36, 241, 245 metabolites, 36, 94, 132, 194, 201, 278 metabolizing, 301 metalloproteinase, 74, 305 metalloproteinases, 32, 72, 73 metals, 4, 18, 194, 260, 264, 304 metformin, 190, 212, 240, 244, 254 methionine, 10, 167, 174, 244, 245, 255 methyl group, 7, 244 methylation, 9, 244 methylprednisolone, 107 MHC, 72, 195, 305 mice, 22, 30, 32, 33, 43, 51, 52, 54, 55, 77, 85, 87, 88, 89, 125, 132, 133, 163, 168, 169, 171, 179, 180, 182, 183, 184, 185, 187, 188, 200, 201, 204, 206, 211, 213, 218, 227, 231, 232, 256, 264, 266, 267, 271, 272, 279, 280, 281, 287, 289, 293, 296, 309, 321 microarray, 107 microcirculatory, 209 microdialysis, 321

342 microenvironment, 4, 46, 168, 177 microfilaments, 128 microglia, 259, 263, 265, 272, 309 microglial, 279, 321 micronutrients, 170, 202, 215 microparticles, 140 microsomes, 108 microtubule, 116, 128, 287 microtubules, 262 microvascular, 40, 109, 200, 201, 204, 205, 206, 209, 213, 221 middle-aged, 269 migration, 26, 28, 29, 33, 37, 67, 69, 70, 71, 73, 77, 86, 116, 146, 150, 166, 238, 252, 266 mild cognitive impairment, 285, 286, 290, 294 milk, 18 mimicking, 171 minerals, 58 MIP, 236 miscarriage, 151 misfolded, 277 misfolding, 231 mitochondria, xii, 15, 18, 20, 24, 38, 40, 57, 76, 96, 108, 121, 123, 124, 131, 164, 174, 180, 181, 194, 196, 207, 223, 228, 230, 232, 249, 253, 255, 259, 260, 264, 279, 280, 281, 289, 295, 305, 306, 310, 319, 322 mitochondrial, 2, 4, 7, 10, 15, 32, 36, 38, 40, 42, 46, 59, 76, 96, 100, 107, 108, 116, 120, 121, 123, 130, 131, 132, 164, 171, 174, 182, 190, 197, 198, 204, 208, 210, 211, 225, 227, 228, 230, 232, 233, 236, 241, 244, 245, 250, 251, 253, 260, 261, 262, 264, 268, 278, 279, 280, 281, 283, 286, 288, 290, 295, 296, 303, 304, 310, 311, 315, 318, 322 mitochondrial abnormalities, 251, 253, 304 mitochondrial damage, 10, 96, 131, 265, 278 mitochondrial DNA, 76, 107, 280, 290, 296 mitochondrial membrane, 10, 40, 76, 264 mitogen, 33, 51, 212 mitogen activated protein kinase, 33 mitogen-activated protein kinase, 51, 212 mitogenesis, 23, 33 mitogenic, 17, 28, 31, 70, 85 mitotic, 260 ML, 18, 57, 86, 105, 131, 156, 177, 179, 181, 185, 247, 251, 293, 294, 318 MMP, 72, 146, 147 MMP-9, 72, 146, 147 MnSOD, 40, 289 modalities, 105, 226

Index model system, 232, 306 modeling, 44, 296 models, viii, xiii, 7, 25, 30, 42, 46, 49, 75, 102, 123, 124, 125, 126, 132, 145, 163, 167, 168, 169, 170, 171, 176, 186, 196, 200, 201, 203, 205, 206, 213, 217, 226, 227, 228, 231, 232, 233, 238, 239, 241, 243, 244, 245, 255, 257, 261, 264, 266, 271, 274, 276, 278, 279, 281, 283, 288, 294, 300, 302, 306, 307, 311, 315, 318, 319 modulation, viii, 25, 27, 33, 37, 44, 59, 70, 96, 100, 104, 107, 124, 132, 166, 168, 178, 203, 265, 266, 310 moieties, 208 molecular biology, 254 molecular changes, 82, 140 molecular mechanisms, 15, 28, 44, 85, 87, 168, 169, 235 molecular oxygen, 5, 11, 34, 37, 38, 39, 40, 75, 76, 228, 229, 230, 306 molecular weight, 263, 277 molecules, vii, ix, 1, 2, 4, 8, 9, 10, 11, 13, 17, 22, 34, 35, 36, 42, 44, 55, 63, 71, 72, 73, 77, 79, 85, 89, 96, 97, 104, 113, 116, 122, 130, 138, 139, 140, 144, 146, 150, 151, 162, 164, 166, 170, 172, 176, 195, 206, 219, 228, 233, 235, 236, 237, 238, 240, 241, 265, 305 monkeys, 302, 312, 314 monoamine, 275 monoamine oxidase, 275 monocyte, 69, 73, 79, 84, 89, 90, 265 monocyte chemoattractant protein, 69, 265 monocyte chemotactic protein, 84 monocytes, ix, 33, 42, 63, 69, 71, 73, 74, 77, 79, 89, 90, 120, 122, 141, 155, 206, 209, 221, 234, 237 monolayer, 27, 36 monomeric, 288 mononuclear cell, 42, 125 mononuclear cells, 42 Moon, 212 morbidity, ix, x, 12, 91, 92, 111, 122, 126, 135, 136, 149, 173, 194, 258, 259 morphological, 119, 141, 226, 228 morphology, 66, 200, 238, 246, 296, 313 morphometric, 129 mortality, viii, ix, x, 12, 15, 46, 60, 63, 64, 65, 80, 90, 91, 111, 112, 126, 128, 135, 136, 160, 173, 177, 190, 200, 258, 259 mortality rate, 112, 160, 259 motion, 294 motor function, 258, 266

Index motor skills, 261 mouse, 30, 56, 73, 84, 163, 168, 171, 185, 200, 201, 204, 213, 217, 218, 232, 241, 266, 288, 289, 290, 292 mouse model, 30, 163, 168, 204, 217, 241, 266, 288, 292 movement, 113, 226, 234 MPTP, 278, 279, 281, 282, 295, 296 MRI, 269 mRNA, 31, 84, 124, 140, 142, 144, 153, 155, 200, 201, 204, 207 MS, 14, 19, 51, 82, 83, 87, 88, 129, 130, 132, 150, 153, 155, 178, 183, 220, 286, 290, 294, 319 mtDNA, 96, 268 MTHFR, 184 multiple factors, 16 multiple sclerosis, 302, 305 multiplication, 71 murine model, 168, 206, 264, 267 murine models, 168, 206, 264 muscle, 13, 19, 21, 23, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 42, 49, 51, 52, 53, 56, 58, 66, 72, 76, 82, 83, 84, 85, 86, 87, 88, 89, 94, 95, 100, 107, 113, 120, 122, 148, 152, 161, 162, 178, 194, 196, 201, 210, 214, 305 muscle cells, 13, 21, 23, 27, 28, 31, 32, 36, 51, 53, 58, 66, 72, 76, 83, 84, 86, 87, 88, 94, 120, 148, 152, 162, 178, 201, 305 muscle contraction, 95 muscle tissue, 122 muscles, 32, 255 Muslim, 109, 156 mutagenic, 8, 9 mutant, 266, 279, 289, 314 mutation, 8, 21, 169 mutations, 7, 8, 9, 21, 161, 162, 227, 261, 277, 278, 279, 296 MV, 51, 156, 181, 190, 218, 251, 316 myelin, 305, 306 myeloid, 72 myeloma, 119 myeloperoxidase, 20, 42, 84, 172 myocardial infarction, 26, 43, 51, 59, 81, 102, 109, 110, 120, 177, 291 myocardial ischemia, 37 myocardial tissue, 104 myocardium, 106, 107 myocyte, 109, 122 myocytes, 109, 162 myofibrillar, 98, 106, 186

343

myofibroblasts, 237, 252 myoglobin, 122, 210 Myoglobin, 122, 130 myometrium, 139 myosin, 72 myricetin, 320

N NA, 30, 34, 47, 54, 86, 247, 316 Na+, 116, 129, 152 N-acety, 22, 43, 47, 59, 61, 62, 92, 99, 101, 105, 125, 133, 180, 189, 201, 205, 239, 241, 243, 244, 254, 311 NAD, 22, 23, 39, 41, 51, 52, 53, 56, 58, 75, 83, 87, 101, 106, 109, 152, 155, 171, 183, 187, 190, 218, 219, 290, 292 NADH, 18, 38, 74, 75, 76, 83, 87, 228 NAS, 231 Nash, 128 National Health Interview Survey, 190 natural, 7, 78, 142, 174, 182, 215, 241, 247, 261, 307, 319, 321 natural food, 174 NC, 57, 60, 288, 289 ND, 23, 258, 261, 264, 266, 267, 276, 280 neck, 162 necrosis, ix, 91, 92, 93, 96, 97, 102, 114, 116, 117, 122, 126, 128, 129, 141, 180, 182, 188, 196, 234, 236, 238, 252, 260, 290 neocortex, 263, 264, 272, 288 neoplastic, 238 neovascularization, 204, 308 nephritis, 119, 130 nephron, 17, 113, 115 nephropathy, xi, 115, 123, 129, 133, 183, 193, 194, 198, 204, 205, 207, 220 nephrosis, 115 nephrotoxic, 112, 117, 118, 123, 127, 131 nephrotoxic drugs, 123 nephrotoxicity, 123, 125, 129, 132, 133 nerve, xiii, 58, 196, 206, 218, 277, 299, 301, 305, 306 nerve cells, 196, 306 nerve conduction velocity, 206 nerve fibers, 306 nerves, 259 nervous system, 33, 170, 211, 265, 292, 315 Netherlands, 274 network, 93, 128, 237

344 neural networks, 163 neuritic plaques, 263 neurobehavioral, 184 neurobiology, 284 neurodegeneration, 258, 259, 277, 278, 288, 309, 312, 313, 321 neurodegenerative, xii, 15, 211, 257, 260, 261, 263, 265, 268, 271, 274, 276, 277, 278, 279, 281, 283, 284, 285, 287, 290, 296, 300, 302, 309 neurodegenerative disease, xii, 15, 257, 283, 285, 290, 296, 302, 309 neurodegenerative diseases, xii, 257, 283, 290, 296, 302, 309 neurodegenerative disorders, 260, 261, 285, 287 neurodegenerative processes, 211, 271, 277, 278, 281, 283 neurofibrillary tangles, 15, 262 neuroimaging, 269 neurological condition, 262 neurological disease, 289, 321 neurological disorder, 276 neuromodulator, 266 neuron death, 14, 15 neuronal apoptosis, 265, 285, 287 neuronal cells, 272, 289 neuronal death, 263, 286 neuronal degeneration, 272, 301 neuronal loss, 258, 262, 277, 279, 321 neuronal plasticity, 271 neurons, 14, 15, 233, 251, 258, 260, 262, 263, 264, 265, 276, 277, 280, 281, 282, 287, 296, 301, 310, 314, 315 neuropathologic changes, 292 neuropathological, 286 neuropathy, xi, xiii, 193, 198, 206, 299, 300, 302, 305, 311, 312, 317 neuroprotection, 281, 284, 303, 314, 319, 320, 322 neuroprotective, 263, 266, 281, 287, 297, 308, 322 neuropsychological assessment, 297 neurotoxic, 263, 264, 278, 281, 282, 283, 288 neurotoxicity, 264, 282, 288, 294, 295, 301, 302 neurotransmission, 302 neurotransmitter, 33, 262, 265, 266, 279, 289, 301 neurotransmitters, 33 neutralization, 240, 316 neutrophil, 3, 5, 6, 7, 20, 38, 42, 116, 153, 183, 212, 252 neutrophils, 7, 13, 71, 77, 116, 122, 129, 140, 141, 198, 202, 234, 237 New York, iii, iv, 131

Index New Zealand, 81 NFT, 262, 263, 264, 265, 267 NF-κB, 235 Ni, 23 nicotinamide, 38, 74, 86, 106, 207, 232, 245, 282, 308 nicotine, 101 Nielsen, 149 nigrostriatal, 277 nitrate, 10, 77 nitric oxide, viii, ix, xi, xii, 2, 19, 20, 21, 22, 23, 25, 26, 45, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 69, 70, 76, 87, 88, 94, 101, 108, 109, 120, 126, 131, 138, 141, 146, 152, 153, 155, 156, 159, 167, 170, 184, 185, 188, 203, 218, 219, 223, 232, 250, 290, 292, 295, 300, 303, 314, 315, 316, 317, 321 nitric oxide (NO), viii, xii, 25, 26, 69, 184, 203, 223, 232, 300 nitric oxide synthase, 19, 21, 22, 23, 45, 53, 54, 56, 57, 60, 61, 69, 70, 76, 87, 88, 94, 101, 108, 109, 120, 126, 138, 141, 152, 156, 219, 232, 250, 290, 292, 314, 317, 321 nitric-oxide synthase, 54 Nitrite, 77 nitrogen, vii, viii, x, 1, 2, 19, 25, 39, 69, 84, 94, 111, 113, 120, 130, 131, 231, 232, 260, 266 nitrosamines, 8 nitrosative stress, 130, 302 nitrous oxide, 279 NK, 81, 152, 156, 252, 288 NMDA, 266 NMDA receptors, 266 N-methyl-D-aspartate, 167 NO, viii, xii, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16, 19, 21, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 38, 39, 41, 43, 44, 45, 48, 50, 57, 59, 61, 70, 71, 74, 76, 77, 80, 100, 120, 121, 126, 131, 145, 148, 166, 167, 171, 184, 200, 203, 204, 205, 213, 218, 219, 223, 232, 269, 300, 302, 304, 309, 310, 315 NO synthase, viii, 5, 26, 29, 32, 34, 38, 49, 57, 59, 204, 218, 232, 309, 310 NO synthases, 5 nodules, 238 non-alcoholic fatty liver, 180, 247, 248, 249, 251 non-enzymatic, 4, 10, 11, 12, 37, 104, 231 non-immunological, 228, 233 non-institutionalized, 273 non-insulin dependent diabetes, 203 non-invasive, 180, 226 non-native, 230

Index non-steroidal anti-inflammatory drugs, 117 non-vascular, 269 norepinephrine, 33, 118 Norfolk, 187 normal, vii, viii, 1, 3, 4, 10, 13, 25, 28, 29, 30, 32, 34, 46, 66, 67, 69, 73, 94, 95, 112, 113, 120, 125, 137, 139, 140, 142, 144, 146, 147, 150, 151, 152, 153, 155, 161, 165, 166, 177, 182, 202, 217, 232, 233, 236, 238, 255, 258, 260, 261, 263, 264, 266, 277, 280, 285, 287, 290, 301, 310, 314, 317, 320 normal aging, 258, 277, 290 normal conditions, vii, 3, 13, 28, 32, 166, 233 normalization, 123, 162, 242 North Africa, 279 North America, 224, 273 NOS, 5, 6, 13, 29, 34, 35, 36, 37, 39, 48, 70, 76, 94, 101, 120, 138, 200, 204, 269, 279, 302, 308, 309 Nrf2, 280 NS, 77, 106, 181, 209, 232, 260 NSAIDs, 117, 119, 165 N-terminal, 279, 287 nuclear, xi, 31, 98, 107, 138, 159, 170, 178, 194, 210, 232, 268, 272, 290 Nuclear factor, 235 Nuclear factor kappa, 235 nuclear magnetic resonance, 210 Nuclear magnetic resonance, 18 nuclear receptors, 178, 272 nuclei, 277 nucleic acid, 119, 197 nucleotides, 39 nucleus, 4, 301, 315 nulliparous, 145 nutrient, 75, 208, 284 nutrients, x, 135, 152, 155, 202, 258, 264, 271, 292, 293, 307 nutrition, 57, 186, 190, 271 nutritional supplements, 273 nuts, 49

O oat, 29 obese, 162, 163, 164, 165, 169, 170, 173, 179, 182, 184, 185, 186, 187, 189, 216, 224, 242, 244, 248, 253 Obese, 231 obese patients, 162, 165, 182 obesity, xii, 65, 136, 160, 161, 162, 163, 164, 166, 168, 169, 170, 172, 176, 177, 178, 179, 180, 183,

345

185, 186, 190, 200, 223, 224, 225, 227, 231, 233, 238, 239, 244, 246, 248, 249, 250, 252, 253 obligate, 311 observations, 160, 166, 168, 174, 196, 201, 305 obstruction, 24, 114, 115, 116, 118, 119, 122 occipital cortex, 262 ocular diseases, 303, 307 ODS, 187 oil, 48, 49, 61, 149, 173, 185, 189, 255 older adults, 190, 294 oligomers, 263, 277, 287 oligonucleotides, 8, 20 olive, 48, 49, 173, 189 olive oil, 48, 49, 173, 189 Omega-3, 311 Oncogene, 211 oncogenes, 9 open angle glaucoma, 16, 300, 311, 312, 313 ophthalmic, 310, 322 optic nerve, xiii, 16, 299, 300, 301, 305, 306, 311, 313, 314, 317, 318 optic neuritis, 320 oral, 44, 47, 48, 59, 60, 90, 103, 124, 125, 133, 187, 203, 204, 210, 218, 244, 245, 255, 277, 310, 311 organ, viii, 15, 16, 17, 25, 26, 27, 39, 47, 104, 112, 115, 121, 137, 140, 164, 197, 206, 207, 271, 280 organelle, 96, 100, 228, 230, 264, 279 organelles, xii, 96, 128, 223, 229 organic, 4, 6, 11, 96, 118, 119, 125 organic compounds, 4 organic peroxides, 11 organic solvent, 118 organic solvents, 118 organism, vii, 17, 94, 235, 318 osteopontin, 172 osteoporosis, 185 ototoxicity, 123, 125, 133 ovariectomized, 188 ovariectomized rat, 188 ovariectomy, 171 ovary, 189 overload, xii, 133, 180, 223, 228, 260, 296 overnutrition, 164 overproduction, 172, 175, 265 overweight, 190, 215, 249, 252 oxalate, 119 oxidants, 2, 69, 73, 74, 84, 87, 88, 89, 119, 122, 153, 199, 302 oxidation, ix, xii, 1, 5, 6, 7, 8, 9, 10, 11, 14, 21, 34, 39, 41, 44, 46, 48, 63, 68, 69, 75, 76, 77, 79, 82,

Index

346 83, 84, 89, 90, 94, 95, 102, 112, 119, 123, 125, 138, 153, 160, 164, 166, 171, 172, 173, 175, 179, 184, 189, 194, 196, 198, 199, 203, 216, 223, 226, 228, 230, 231, 233, 243, 250, 254, 255, 260, 265, 266, 267, 268, 269, 278, 279, 280, 281, 285, 290, 308, 309 oxidative damage, vii, viii, xi, xiii, 1, 15, 16, 20, 25, 46, 96, 100, 102, 109, 159, 160, 171, 181, 194, 199, 203, 212, 233, 241, 244, 246, 264, 265, 270, 272, 276, 285, 289, 290, 292, 293, 296, 299, 300, 310 oxidative reaction, 241 oxide, ix, xi, 2, 5, 13, 19, 20, 21, 22, 23, 29, 33, 41, 45, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 69, 70, 76, 77, 85, 87, 88, 94, 101, 108, 109, 120, 126, 131, 138, 141, 146, 152, 153, 155, 156, 159, 167, 170, 181, 184, 185, 188, 218, 219, 232, 250, 275, 279, 289, 290, 292, 295, 302, 304, 312, 314, 315, 316, 317, 321 oxygen, vii, viii, x, 1, 2, 3, 5, 6, 11, 12, 17, 18, 21, 22, 25, 26, 33, 37, 38, 39, 40, 41, 51, 55, 57, 58, 63, 64, 74, 75, 76, 77, 84, 86, 90, 92, 94, 96, 102, 103, 111, 115, 116, 117, 120, 121, 122, 123, 126, 128, 130, 131, 132, 137, 139, 142, 146, 152, 163, 175, 201, 228, 229, 230, 238, 249, 259, 260, 303, 304, 306, 308, 310, 319 Oxygen, 3, 24, 130, 132, 150 oxygen consumption, 94, 115, 128, 260 oxygenation, 21 oxyhemoglobin, 121 oxyradicals, 20

P p53, 9, 21, 235, 305 PA, 87, 108, 151, 157, 178, 182, 185, 188, 208, 252, 286, 291, 293, 295, 315, 318 pacemaker, 93 pacemakers, 93 pacing, 102, 103, 105 PAI-1, 123, 143, 144 pain, 137 pancreas, 32, 203, 227 pancreatic, xi, 161, 176, 184, 193, 194, 195, 196, 199, 201, 205, 207, 209, 210, 211, 212, 213, 214, 217, 250 pancreatic acinar cell, 184 pancreatic islet, 195, 199, 209, 213, 217 pantothenic acid, 243 paracrine, 29, 33, 235, 237

paradox, 3, 57, 305 paradoxical, 28, 29 parameter, 14, 242, 311 parenchyma, 237 parenchymal, 234, 236, 237, 252 parenchymal cell, 234, 252 parenteral, 125 parietal lobe, 268 Parkinson, vii, xii, 15, 257, 261, 266, 276, 277, 278, 280, 281, 283, 284, 292, 294, 295, 296, 297 Parkinson disease, vii, 266, 280, 295, 296 Parkinsonism, 276, 282, 296, 297 particles, 57, 67, 68, 82, 166, 189, 226, 236, 238 paternity, 149 pathogenic, xi, xiii, 16, 92, 93, 120, 159, 160, 165, 182, 189, 276, 278, 295, 299, 300, 304 pathogens, 2, 6, 48, 234, 235 pathology, 16, 27, 64, 76, 92, 136, 225, 228, 231, 240, 241, 243, 254, 258, 264, 267, 271, 277, 287, 289, 292, 293, 294 pathophysiological, viii, xiii, 2, 4, 7, 10, 12, 14, 17, 25, 26, 28, 36, 37, 42, 64, 75, 76, 87, 92, 94, 113, 114, 115, 117, 137, 140, 143, 146, 148, 206, 257, 302 Pathophysiological, 7, 155 pathophysiological mechanisms, xiii, 143, 257 pathophysiology, vii, ix, x, xii, xiii, 27, 30, 41, 50, 51, 55, 56, 64, 68, 70, 73, 74, 75, 78, 81, 91, 92, 105, 111, 112, 116, 122, 123, 126, 127, 136, 137, 140, 141, 149, 150, 160, 168, 178, 186, 224, 225, 250, 251, 257, 276, 288, 299, 300, 301, 303, 310, 312 pathways, xi, xii, xiii, 4, 7, 12, 14, 16, 17, 28, 36, 45, 46, 55, 58, 94, 96, 103, 109, 123, 132, 156, 159, 161, 165, 166, 167, 176, 199, 200, 206, 207, 208, 210, 212, 220, 223, 224, 225, 226, 227, 229, 230, 231, 234, 246, 253, 268, 269, 278, 299, 306, 311 pattern recognition, 71 PCR, 96 PD, 15, 60, 85, 130, 133, 180, 276, 277, 278, 279, 280, 281, 282, 283, 284, 296 PE, x, 83, 129, 131, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 179, 290 pediatric, 242, 253 pelvis, 114 Pennsylvania, 273 pentamidine, 118 peptidase, 228, 241

Index peptide, 15, 30, 32, 36, 49, 113, 261, 263, 265, 287, 309 peptides, 28, 31, 259, 262, 263, 264, 272, 283 perceptions, 105 perfusion, 15, 16, 114, 115, 132, 137, 139, 140, 141, 143, 288, 304, 305, 312, 317 pericarditis, 114 pericytes, 237, 305 perinatal, 136, 137, 156 Peripheral, 285, 296 peripheral blood, 196, 267, 268, 280 peripheral nervous system, 258 peripheral neuropathy, 114 peripheral vascular disease, 22 peritoneal, 84, 137 peritoneal cavity, 137 permeability, 67, 117, 136, 148, 164, 165, 166, 182, 234, 264, 301, 310 peroxidation, x, 9, 10, 14, 21, 43, 58, 116, 135, 142, 144, 152, 164, 174, 198, 203, 208, 213, 226, 231, 241, 266, 303, 307, 308, 312 peroxide, 4, 41, 94, 132, 152, 173, 194, 199, 304, 316 peroxisomes, 40, 132, 164, 181 peroxynitrite, ix, 2, 15, 21, 22, 35, 37, 38, 39, 42, 46, 54, 59, 63, 69, 70, 76, 85, 88, 103, 105, 121, 141, 142, 205, 217, 232, 270, 302, 304, 315 perturbation, 38, 171 perturbations, 160, 163 PF, 18, 115, 132, 291 PG, 105, 149, 220, 295, 311, 318 pH, 152 phagocyte, 52 phagocytic, 38, 74, 120, 122 phagocytosis, 236, 238 pharmaceutical, 203 pharmacodynamics, 215 pharmacokinetics, 44, 59 pharmacological, 12, 31, 92, 125, 200, 205, 208, 240, 245, 311 pharmacological treatment, 92, 311 pharmacology, 48, 60, 156, 250, 285 pharmacotherapy, 33, 178 phenol, 107, 320 phenolic, 48, 173, 308, 309 phenolic acids, 48 phenolic compounds, 308 phenotype, 73, 86, 139, 150, 161, 169, 183, 266, 271, 272, 276, 279, 290 phenotypes, 169

347

phenotypic, 73, 169, 185 phenytoin, 119 Philadelphia, 128 phosphatases, 120 phosphate, 32, 38, 41, 74, 86, 101, 106, 113, 197, 206, 207, 308 Phosphate, 322 phosphatidic acid, 33 phosphatidylcholine, 33, 244, 245 phospholipase C, 29, 51 phospholipids, 9, 68, 83, 116, 120, 142 phosphorylation, 10, 29, 76, 95, 97, 120, 121, 123, 145, 155, 177, 182, 187, 196, 210, 264, 272, 287, 310 photoreceptor, 308 photoreceptor cells, 308 photoreceptors, 309, 320 physical activity, 242 physical properties, 255 physicians, 176, 248 Physicians, 259 physiological, vii, viii, 1, 2, 3, 7, 12, 13, 25, 27, 28, 31, 33, 36, 50, 89, 94, 115, 121, 137, 139, 140, 146, 148, 172, 217, 227, 230, 232, 233, 237, 245, 277, 285, 310 physiology, 55, 56, 112, 168, 263, 280, 285, 288 phytoestrogens, 245 PI3K, 252 pig, 109, 145, 289, 313 pigments, 79, 202 pigs, 102 pilot study, 105, 189, 216, 221, 242, 243, 247, 253, 256, 282, 317 pineal, 272 pioglitazone, 242, 247 PKC, 31, 320 PL, 20, 105, 108, 153, 216, 219, 250, 284, 312, 314, 315 placebo, 47, 59, 60, 79, 99, 103, 105, 125, 146, 156, 190, 203, 242, 243, 244, 245, 247, 255, 256, 271, 272, 275, 276, 281 placenta, 16, 137, 138, 139, 140, 141, 142, 145, 146, 147, 150, 151, 153, 154, 156 placental, x, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156 plants, 48, 174, 240 plaque, 64, 66, 67, 72, 73, 74, 263, 267, 271, 272, 289, 292

348 plaques, 67, 69, 72, 99, 166, 198, 259, 263, 265, 283, 301 plasma, x, 11, 14, 32, 34, 37, 42, 44, 46, 55, 58, 59, 67, 68, 69, 79, 83, 84, 112, 113, 115, 116, 118, 119, 122, 130, 132, 135, 142, 143, 144, 152, 153, 155, 160, 163, 165, 169, 170, 171, 173, 174, 182, 183, 187, 188, 196, 202, 203, 215, 216, 232, 233, 234, 241, 244, 245, 267, 268, 269, 291, 292, 305, 306, 315, 317, 320, 322 plasma levels, 14, 142, 144, 155, 165, 182, 188, 245, 267, 269, 291, 317, 322 plasma membrane, 34, 116, 232, 306, 315 plasmids, 9 plasminogen, 72, 150 plastic, 266 plasticity, 263, 271, 291 platelet, 13, 27, 33, 58, 67, 69, 70, 71, 72, 73, 79, 86, 89, 100, 137, 145, 166, 238, 310, 321 Platelet, 137 platelet aggregation, 13, 33, 67, 70, 71, 73, 100, 145, 166, 310, 321 platelet count, 137 platelet derived growth factor, 69 platelets, 42, 67, 73, 150 play, viii, xi, xiii, 11, 15, 16, 17, 25, 26, 32, 33, 36, 39, 48, 71, 78, 94, 102, 104, 112, 120, 121, 124, 126, 144, 145, 147, 163, 172, 193, 198, 202, 207, 228, 236, 238, 241, 268, 270, 280, 299, 300, 303, 311 plethysmography, 70 plexus, 301 PM, 55, 60, 83, 86, 87, 128, 132, 149, 152, 180, 182, 183, 214, 290, 291, 296 PN, 157, 217 pneumonia, 118, 259 PO, 213, 214 POAG, 16, 300, 303, 304, 306, 311, 322 poisoning, 119 Poland, 209 polarity, 116, 117, 128 polycyclic aromatic hydrocarbon, 231 polygenic, 168 polymerization, 262 polymorphism, 184, 270, 292 polymorphisms, 303 polymorphonuclear, 37, 42, 120 polymorphonuclear cells, 37 polyphenolic compounds, 49, 174, 308 polyphenols, 48, 61, 62, 126, 172, 190, 282, 308, 309, 319, 320, 321

Index Polyphenols, 48, 320 polyunsaturated fat, 9, 11, 21, 59, 173, 190, 227, 249, 260, 284, 308 polyunsaturated fatty acid, 9, 11, 21, 59, 173, 190, 227, 249, 260, 284, 308 polyunsaturated fatty acids, 11, 21, 59, 173, 190, 227, 260, 284 pomegranate, 185, 216 pools, 66 poor, x, 92, 93, 104, 135, 143, 148, 263, 271, 273 population, ix, xii, xiii, 10, 12, 50, 62, 92, 104, 111, 146, 149, 160, 179, 186, 223, 224, 248, 257, 258, 261, 269, 270, 274, 276, 280, 282, 286, 300, 307 pores, 236 positive correlation, 101, 202 positive feedback, 7, 96, 229 postmenopausal, 189 postmenopausal women, 189 postmortem, 278 postoperative, ix, 91, 92, 97, 104, 105, 107, 108 postoperative outcome, 105 post-transcriptional regulation, 95 post-translational, 145, 214 potassium, 49, 114, 117 potatoes, 49 powder, 271 power, 9, 56, 92, 215, 305 PPP, 46, 60 precipitation, 118, 230 preclinical, xi, 193, 207, 208 preconditioning, vii, 106 prediction, 65, 81, 86, 149, 154 predictors, x, 135 preeclampsia, vii, 1, 12, 15, 16, 24, 31, 52, 60, 149, 150, 151, 152, 153, 154, 155, 156, 157, 268 pre-eclampsia, 136, 138, 140, 146i, 147, 149, 150, 151, 152, 153, 154, 155, 156 pre-existing, 118, 136, 143 pregnancy, x, 15, 16, 28, 135, 136, 137, 139, 140, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 244 pregnant, 136, 140, 143, 145, 146, 148, 152, 153, 154, 156 pregnant women, 136, 140, 145, 146, 148, 152, 153, 154, 156 presenilin 1, 266 pressure, viii, xiii, 3, 13, 16, 25, 26, 27, 28, 30, 31, 33, 34, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 50, 52, 56, 58, 59, 60, 61, 62, 65, 74, 81, 109, 113, 115, 116, 136, 168, 170, 171, 172, 175, 176, 237,

Index 259, 299, 300, 301, 303, 304, 306, 308, 310, 311, 312, 313, 314, 315, 317, 318, 319, 320, 322 prevention, x, 15, 16, 27, 43, 46, 49, 50, 52, 57, 58, 59, 60, 64, 65, 80, 89, 90, 99, 102, 103, 105, 108, 110, 111, 112, 123, 124, 125, 126, 135, 143, 144, 145, 148, 149, 155, 171, 172, 175, 176, 201, 214, 215, 240, 241, 246, 254, 261, 269, 270, 271, 301, 310, 312 preventive, x, 47, 104, 111, 126, 190, 194, 202 primary open-angle glaucoma, 312, 313, 315, 316, 317 primary open-angle glaucoma (POAG), 313 primates, 53, 281 pro-apoptotic protein, 235 probability, 34, 70, 275, 305 procoagulant, 148, 160 producers, 33, 120 pro-fibrotic, 93, 97 progenitor cells, 125 progenitors, 313 prognosis, 13, 149, 286 program, 147, 206 proinflammatory, xi, 37, 41, 96, 193, 195, 200, 206, 209, 225, 234 pro-inflammatory, 26, 168, 235, 236, 237, 259, 265, 269 pro-inflammatory response, 265 proliferation, ix, 13, 23, 28, 29, 33, 37, 51, 63, 64, 67, 70, 71, 73, 74, 77, 79, 80, 85, 89, 145, 148, 201, 232, 238, 245, 252, 260, 262, 320 promoter, 200, 210, 227 pro-oxidant, vii, 2, 35, 96, 125, 133, 141, 233, 260, 261, 278, 304, 316 propagation, 9, 41, 92, 160, 234, 236, 240, 242 propane, 142 property, 100, 166, 219, 244 prophylactic, ix, 91, 126, 156 prophylaxis, 104, 129, 155, 174, 203, 216 prostaglandin, 7, 21, 31, 35, 45, 51, 143, 171, 199, 266, 268, 289, 317 prostaglandins, 28, 113, 234 prostanoids, 59, 101, 109, 201 protease inhibitors, 162 proteases, 72, 73, 75, 116, 120, 263 Proteasome, 279, 295 protection, ix, xi, 18, 19, 34, 49, 63, 84, 108, 124, 125, 159, 169, 171, 181, 193, 204, 208, 214, 217, 219, 254, 260, 265, 268, 280, 282, 303, 307, 313, 316, 319, 322 protective mechanisms, 208

349

protective role, 28, 71, 79, 80, 207, 281 proteic, 4 protein, 2, 7, 9, 10, 11, 14, 15, 18, 21, 31, 32, 34, 41, 58, 68, 69, 72, 77, 82, 83, 84, 89, 95, 96, 97, 112, 113, 119, 123, 132, 141, 142, 144, 145, 153, 155, 165, 171, 173, 179, 181, 182, 184, 185, 194, 195, 198, 202, 204, 206, 214, 217, 227, 230, 232, 233, 234, 235, 236, 255, 260, 261, 262, 263, 265, 266, 267, 268, 270, 278, 279, 280, 285, 287, 290, 305, 307, 310, 314 protein disulfide isomerase, 230 protein folding, 230 protein kinase C, 31, 89, 95, 123, 194, 206 protein kinase C (PKC), 31 protein kinases, 230 protein misfolding, 231 protein oxidation, 7, 10, 69, 83, 112, 153, 255, 265, 266, 267, 278, 279, 285, 290 protein synthesis, 230 proteinase, 252 proteins, xi, 4, 8, 10, 15, 17, 20, 21, 27, 28, 40, 41, 44, 48, 60, 71, 72, 76, 77, 86, 95, 96, 97, 104, 116, 119, 123, 130, 136, 140, 141, 142, 168, 172, 177, 184, 193, 194, 197, 198, 203, 230, 235, 260, 261, 262, 265, 268, 271, 272, 277, 280, 281, 285, 287, 289, 290, 306, 310, 313, 315 Proteins, 267 proteinuria, x, 119, 135, 136, 138, 148, 154, 205 proteoglycans, 67, 72, 73 proteolysis, 40, 263 proteolytic enzyme, 263 proteomics, 290 protocol, 169, 307 protocols, 8, 104 protons, 6 proximal tubule cells, 116 pseudo, 147 PT, 82, 154, 156, 181 public, xiii, 175, 194, 299 public health, xiii, 175, 194, 299 PUFA, 9, 11, 14, 17, 260, 268, 271, 308, 311 pulmonary arteries, 93 pulse, 46, 70 purification, 2 purines, 5, 75 pyrene, 202 pyridoxamine, 206, 220 pyrimidine, 7

Index

350

Q quail, 302, 314 quality control, 278 quality of life, 261 quercetin, 48, 62, 107 Quercetin, 62, 185 quinine, 119 quinone, 41, 100, 108 quinones, 278, 281

R RA, 22, 54, 57, 59, 60, 61, 83, 90, 106, 128, 130, 131, 132, 156, 178, 179, 210, 213, 215, 249, 256, 272, 288, 290, 313, 314, 321 race, 89, 273 radiation, 2, 40, 48, 303 radical formation, 39, 119, 123 radical mechanism, 21 radiologists, 304 RAGE, 197, 198, 206, 207, 208, 211, 212 rail, 21 Ramadan, 177 Ramipril, 214 random, 217, 275 random assignment, 275 range, 9, 18, 27, 33, 71, 73, 205, 206, 208, 231, 238, 244, 261 RAS, 30 rat, 22, 23, 32, 52, 53, 56, 62, 73, 83, 102, 108, 109, 121, 124, 126, 131, 132, 134, 169, 171, 174, 177, 180, 182, 184, 185, 186, 187, 188, 200, 204, 205, 212, 219, 220, 221, 233, 245, 252, 253, 254, 255, 289, 302, 306, 312, 313, 314, 315, 318, 319, 321 rating scale, 262, 282 rats, 20, 23, 30, 39, 43, 47, 48, 51, 54, 56, 58, 60, 61, 62, 102, 108, 109, 125, 126, 131, 133, 134, 145, 148, 155, 156, 169, 170, 172, 180, 181, 184, 185, 186, 187, 188, 190, 200, 201, 204, 206, 213, 214, 217, 218, 219, 221, 243, 249, 251, 253, 254, 255, 269, 281, 302, 307, 314, 315, 316, 319, 322 RB, 81, 105, 182, 190, 216, 284, 285, 291, 295 RC, 18, 133, 210, 215, 286, 288, 294, 297, 316, 321 reactant, 14, 203 reactants, 72 reaction rate, 44 reactive nitrogen, viii, 25, 120, 130, 232

reactive oxygen, vii, ix, x, xi, xii, xiii, 1, 2, 21, 26, 36, 53, 55, 61, 78, 91, 92, 98, 104, 111, 112, 127, 132, 137, 138, 140, 147, 151, 170, 171, 179, 183, 184, 193, 210, 211, 212, 223, 251, 259, 285, 294, 299, 318 reactive oxygen species, vii, ix, xi, xii, xiii, 1, 2, 21, 26, 36, 53, 55, 61, 78, 91, 92, 98, 104, 112, 127, 132, 137, 138, 140, 147, 151, 170, 171, 179, 183, 184, 193, 210, 211, 212, 223, 251, 259, 285, 294, 299, 318 Reactive Oxygen Species, 7, 12, 37, 141, 264, 278 reactive oxygen species (ROS), vii, ix, xi, xii, xiii, 2, 26, 78, 91, 92, 104, 112, 127, 137, 140, 171, 193, 223, 299 reactivity, 4, 16, 35, 42, 43, 69, 79, 138, 142, 184, 218, 241, 254 reagents, 163 reality, 97, 106 reasoning, 261 receptive field, 319 receptor agonist, 52, 152 receptors, xi, 13, 29, 30, 31, 33, 45, 52, 53, 68, 69, 71, 73, 83, 141, 178, 182, 193, 197, 201, 206, 237, 238, 252, 258, 265, 266, 272, 302, 314 recognition, 21, 71, 83, 195, 259 reconcile, 50 recoverin, 18 recovery, 311 recurrence, 108, 109, 149 recycling, 100, 166, 302, 303 red blood cell, 113, 199, 212 red blood cells, 212 red meat, 50 red wine, 24, 48, 50, 61, 62, 126, 133, 171, 309, 320, 321 redistribution, 128 redox, viii, xi, xii, 1, 18, 25, 28, 31, 35, 37, 43, 44, 46, 54, 55, 58, 87, 96, 106, 107, 159, 164, 168, 171, 174, 177, 188, 212, 224, 227, 232, 235, 250, 261, 265, 266, 268, 289, 290, 303, 316 Redox, 19, 20, 51, 54, 55, 58, 85, 86, 108, 208, 212, 250, 290, 295 reducing sugars, 197, 198 redundancy, 221 reflection, 96, 103 refractoriness, 164 refractory, 93, 95, 98, 103 regenerate, 44, 244 regeneration, 96, 177, 229, 234, 235, 237, 313 regional, 115, 150, 290, 306

Index Registry, 297 regression, 49 regular, 185 regulation, viii, x, 6, 22, 23, 24, 26, 27, 28, 30, 34, 35, 37, 40, 43, 44, 45, 46, 53, 54, 55, 56, 58, 59, 66, 70, 71, 85, 86, 88, 92, 95, 96, 100, 101, 109, 113, 131, 136, 141, 145, 146, 147, 148, 150, 152, 155, 160, 163, 168, 170, 173, 174, 180, 181, 182, 183, 187, 200, 201, 205, 207, 212, 219, 227, 232, 237, 249, 250, 258, 265, 266, 301, 306, 312, 315, 316, 318 reinforcement, ix, 18, 46, 91, 101, 104 rejection, 121 relationship, vii, 1, 13, 23, 44, 166, 170, 215, 265, 271, 274, 281, 283, 304 relationships, 306 relaxation, 19, 29, 31, 48, 53, 54, 70, 85, 95, 131, 204, 213, 235 relevance, xii, 17, 22, 65, 66, 87, 103, 104, 113, 123, 126, 196, 208, 223, 277, 300, 306 reliability, 143 remethylation, 167 remodeling, viii, ix, x, xiii, 16, 25, 28, 29, 30, 32, 34, 37, 43, 49, 53, 61, 74, 91, 92, 93, 94, 95, 96, 97, 102, 104, 105, 106, 107, 108, 109, 135, 139, 144, 146, 157, 164, 299, 308, 311 remodelling, 54, 106 renal, vii, ix, x, 11, 12, 13, 17, 22, 23, 24, 30, 32, 41, 44, 47, 53, 58, 60, 61, 81, 94, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 137, 169, 172, 198, 202, 204, 205, 206, 208, 211, 213, 217, 218, 220, 221, 267, 269 renal disease, 17, 22, 24, 61, 81, 114, 119, 123, 124, 125, 130, 202, 220 renal dysfunction, 47, 61, 117, 118, 122, 130 renal epithelial cells, 128, 130 renal failure, ix, 111, 112, 114, 118, 119, 120, 126, 127, 128, 129, 130, 133, 211 renal function, ix, 111, 112, 113, 117, 122, 126, 128, 129, 131, 137, 170, 204, 217, 269 renal hemodynamics, 32, 113 renal medulla, 129 renal replacement therapy, 112 renin, 30, 47, 49, 94, 106, 113, 166, 233, 237, 238 renin-angiotensin system, 94, 233, 237 rennin angiotensin system, 220 reoxygenation, 75, 121, 131 repair, 2, 13, 21, 211, 230, 251, 265, 289 reparation, 9, 229, 230, 234, 235

351

reperfusion, 3, 7, 22, 92, 94, 95, 102, 103, 104, 110, 120, 121, 124, 126, 128, 130, 131, 132, 134, 137, 146, 151, 181, 182, 184, 285, 303, 305, 306, 310, 315, 317 replication, 8, 9 reproduction, 283 reserves, 225, 241 reservoirs, 279 residues, 9, 10, 32, 40, 77, 197 resistance, xi, xii, xiii, 14, 15, 37, 42, 53, 58, 61, 136, 139, 143, 159, 160, 161, 162, 163, 164, 166, 168, 169, 170, 172, 176, 178, 179, 180, 182, 185, 186, 188, 189, 190, 196, 202, 203, 208, 210, 215, 216, 223, 224, 225, 227, 233, 237, 238, 239, 240, 246, 249, 250, 251, 252, 270, 299, 301, 304, 306, 320 resistivity, 310 resolution, 54, 139 resources, xiii, 143, 257 respiration, 2, 7, 10, 17, 123, 228 respiratory, 4, 6, 7, 32, 41, 42, 87, 100, 120, 155, 174, 228, 229, 279 responsiveness, 36, 51, 161, 201, 214 Resveratrol, 49, 134, 188, 321 retention, 82, 84, 94, 164, 206 reticulum, xii, 49, 95, 106, 168, 184, 186, 223, 228, 250 retina, 198, 301, 303, 305, 306, 309, 310, 313, 314, 316, 317, 318, 321 retinal disease, 309 retinoic acid, 283 retinol, 143, 154, 203 retinopathy, 194, 205, 207, 220 RF, 19, 53, 252, 313, 315, 321 rhabdomyolysis, 17, 122, 126, 129, 134 Rhabdomyolysis, 119, 122, 130, 131 rhythm, ix, 91, 92, 93, 96, 97, 104 rigidity, 237 risk, ix, x, xi, xiii, 12, 13, 14, 17, 26, 44, 51, 58, 64, 65, 68, 70, 72, 74, 79, 80, 81, 85, 86, 88, 92, 94, 103, 105, 107, 108, 111, 112, 117, 126, 128, 135, 137, 140, 143, 144, 145, 146, 149, 154, 156, 159, 160, 161, 162, 164, 170, 172, 173, 174, 175, 176, 177, 180, 186, 189, 190, 199, 200, 209, 216, 247, 255, 258, 261, 268, 269, 270, 271, 273, 274, 275, 280, 282, 283, 291, 292, 293, 294, 296, 297, 299, 300, 305, 306, 308, 320, 321 risk assessment, 149, 154

Index

352 risk factors, ix, x, xi, 13, 51, 64, 65, 70, 74, 81, 85, 92, 94, 111, 128, 135, 143, 159, 160, 164, 173, 174, 176, 180, 186, 190, 209, 247, 270, 273, 300 risks, 48, 156, 216, 291 RNA, 52, 260, 265 rodent, 125, 266, 306 rodents, 163, 170, 231 rosiglitazone, 215 RP, 52, 72, 150, 151, 152, 153, 178, 180, 209, 213, 214, 220, 291 rural, 273, 293 Rutherford, 149

S SA, 24, 53, 82, 85, 107, 154, 157, 184, 190, 215, 250, 253, 291, 293, 320 Saccharomyces cerevisiae, 188 safety, 220, 255, 322 saline, 118, 129, 306, 307 salt, 32, 47, 56, 58, 61, 118, 206 sample, 153, 202, 273, 293 saponin, 48 sarcoidosis, 119 saturated fat, 228, 233 saturated fatty acids, 228 scavenger, 10, 68, 69, 71, 73, 83, 100, 121, 125, 126, 174, 204, 205, 206, 219, 265, 288, 320 schema, 101 Schiff, 197 Schiff base, 197 schizophrenia, 266 Schmid, 55, 56 scientific community, 258 sclerosis, 204, 218 scores, 274, 281 scurvy, 29, 79 SD, 84, 109, 186 SE, 52, 83, 85, 154, 251, 288, 292, 296 seafood, 174 search, 92, 140 searching, 273 secrete, 146, 157, 161, 226, 236, 237, 238 secretion, xi, 29, 32, 72, 73, 86, 113, 142, 145, 150, 162, 163, 178, 181, 184, 193, 194, 197, 200, 208, 211, 219, 226, 227, 233, 236, 243, 315 security, 259 seed, 185 seeds, 49 selectivity, 184

selenium, 187, 188, 190, 202, 215, 273 self-report, 273 senescence, 12 senile, 15, 262 senile plaques, 15, 262 sensing, 179, 265 sensitivity, 97, 162, 169, 170, 176, 179, 187, 211, 219, 259, 267, 280, 289, 295 sensors, xi, 159 sepsis, 20, 22, 112, 117, 118, 259, 267 series, 76, 81, 86, 142, 170, 195, 207, 209, 235 serine, 279 serotonin, 35 serum, 19, 23, 57, 94, 97, 99, 113, 125, 126, 136, 137, 142, 143, 149, 152, 153, 154, 155, 160, 163, 164, 165, 172, 174, 202, 203, 204, 206, 216, 243, 245, 273, 280 serum albumin, 19, 202 services, iv severity, xiii, 16, 66, 72, 142, 145, 196, 263, 275, 278, 299 sex, 273, 292 SH, 9, 11, 107, 124, 125, 176, 181, 182, 200, 219, 230, 244, 247, 251, 254, 282, 293 shape, 313 shares, 69, 266 sharing, 275, 276, 278 shear, 13, 26, 27, 28, 32, 34, 39, 56, 69, 70, 71, 73, 75, 83, 87 shock, 20, 72, 86, 115, 129, 306, 315 short period, 199 short-term, 28, 44, 47, 202, 204, 242, 312 Short-term, 48 shoulder, 67, 162 SI, 215 sign, 113 signal transduction, 46, 164, 196, 199, 232, 258 signaling, vii, viii, 2, 7, 20, 23, 25, 26, 29, 30, 35, 36, 37, 43, 52, 55, 58, 62, 87, 106, 109, 184, 186, 194, 196, 206, 208, 210, 212, 214, 230, 250, 252, 269, 288, 304, 314, 320 signaling pathway, 2, 7, 30, 109, 194, 196, 206, 208, 210, 212, 214, 320 signaling pathways, 2, 7, 30, 109, 194, 196, 206, 210, 212 signalling, 51, 107, 289, 291, 316 signals, 18, 26, 38, 105, 199, 236, 237 signs, 114, 148, 196, 277, 305 Singapore, 297 sinus, 93, 96

Index sinus rhythm, 96 siRNA, 30 sites, 4, 7, 9, 30, 41, 67, 73, 74, 116, 125, 160, 162, 164, 198, 265, 268, 275, 276, 289 skeletal muscle, 161, 178 skills, 261 skin, 320 sleep, 306, 318 sleep apnea, 306, 318 small intestine, 7 smoke, 211 smokers, 13, 39, 57, 79, 80, 90, 198, 211, 274 smoking, 65, 70, 89, 90, 107, 176 smooth muscle, 13, 19, 21, 23, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 42, 49, 51, 52, 53, 56, 58, 66, 72, 76, 82, 83, 84, 85, 86, 87, 88, 89, 94, 100, 120, 148, 152, 178, 201, 214, 305 smooth muscle cells, 13, 21, 23, 27, 28, 31, 32, 36, 51, 53, 58, 66, 72, 76, 83, 84, 86, 87, 88, 94, 120, 148, 152, 178, 201, 305 SNpc, 277, 278, 279, 280, 281 social security, 259 SOD, 2, 3, 11, 15, 16, 40, 42, 44, 75, 101, 121, 124, 125, 132, 142, 144, 204, 232, 265, 267, 269, 271, 272, 280, 306 SOD1, 204 sodium, 30, 49, 94, 102, 109, 114, 115, 116, 117, 118, 129, 172, 309, 320 soil, 189 solvents, 231 somatostatin, 32 sorbitol, 194, 203, 216 sounds, 283 soy, 203, 245 soybean, 255 soybeans, 241, 245 SP, 19, 22, 130, 183, 186, 262, 263, 265, 267, 272, 290, 316 spasticity, 277 spatial, 166, 269, 272, 291 spatial learning, 269, 272, 291 specialized cells, 230 species, vii, viii, ix, x, xi, xii, xiii, 1, 2, 4, 5, 9, 11, 12, 14, 17, 21, 25, 26, 36, 44, 51, 53, 55, 57, 58, 61, 63, 64, 69, 78, 79, 86, 91, 92, 98, 104, 111, 112, 120, 123, 126, 127, 130, 132, 137, 138, 140, 147, 151, 163, 170, 171, 175, 179, 183, 184, 193, 210, 211, 212, 223, 229, 232, 233, 235, 237, 238, 240, 241, 246, 249, 251, 259, 260, 263, 266, 285, 294, 299, 306, 308, 318, 319

353

specificity, 39, 77, 259, 267 spectrum, xii, 11, 20, 69, 223, 224, 247, 248 speed, 17 sphingolipids, 232 sphingosine, 232 spin, 18, 183, 200 spinach, 174 spinal cord, 33, 266 sporadic, 15, 258, 261, 270, 277, 279, 280, 283 sprain, 202 SR, 19, 20, 21, 56, 88, 95, 177, 178, 211, 220, 221, 248, 314 stability, 21, 99, 267 stabilization, 103 stages, x, xiii, 3, 4, 9, 12, 43, 80, 83, 135, 143, 147, 180, 237, 257, 259, 261, 264, 265, 266, 267, 268, 321 standard deviation, 262 standards, 275 statin, 105, 271 statins, 65, 81, 92, 99, 107, 119, 205, 240, 270, 292 Statins, 99, 108, 270 statistics, 224 Steatosis, 163, 226, 227 stellate cells, 182, 228, 237, 238, 251, 252 Stellate cells, 238 steroid, 231, 306, 318 Steroid, 306 steroids, 171 stiffness, xi, 60, 193, 198, 321 stimulant, 212 stimulus, 38, 70, 120, 141, 161, 234 storage, xi, 125, 159, 161, 162, 168, 173 strain, 168, 169, 316 strains, 169, 170, 185 strategies, ix, xii, 37, 43, 91, 92, 112, 123, 125, 144, 176, 183, 208, 218, 224, 240, 246, 259, 261 strength, 148, 261 stress-related, vii, xi, 14, 47, 75, 193, 202, 243, 251 striatum, 266 stroke, 12, 13, 37, 43, 64, 92, 137, 170, 173, 190, 315 structural changes, 41 structural protein, 141 Subcellular, 131 substances, xii, 10, 14, 27, 31, 42, 114, 118, 120, 140, 187, 203, 208, 224, 234, 236, 240, 241, 246, 260, 282, 305 substantia nigra pars compacta, (SNpc), 277, 269 substrates, 77, 231

354 sucrose, 180, 190 suffering, 68, 309 sugar, 7, 9, 41, 165 sugars, 7, 49, 197, 198, 210 suicide, 8, 316 sulfonamides, 118, 119 sulphate, 137 Sun, 59, 186, 249 superoxide, viii, xiii, 2, 4, 5, 11, 18, 19, 22, 23, 25, 26, 28, 30, 32, 35, 37, 38, 39, 40, 42, 43, 44, 46, 47, 48, 50, 55, 56, 57, 58, 59, 62, 69, 75, 83, 87, 92, 94, 95, 100, 103, 120, 121, 124, 130, 132, 139, 140, 141, 142, 145, 152, 153, 172, 176, 183, 200, 201, 203, 205, 210, 217, 228, 229, 231, 232, 234, 254, 260, 265, 279, 288, 290, 293, 299, 306, 308, 314, 316 Superoxide, 11, 18, 19, 35, 37, 39, 40, 42, 59, 60, 106, 123, 131, 132, 183, 189 superoxide dismutase, xiii, 2, 18, 19, 40, 75, 92, 121, 124, 130, 139, 142, 152, 153, 172, 200, 203, 205, 217, 232, 234, 265, 288, 293, 299, 316 supplemental, 47, 274 supplements, 11, 44, 46, 59, 90, 173, 203, 216, 248, 273, 274, 283, 284, 293, 294, 310 supply, 117, 208, 264 suppression, 70, 194, 196, 199, 210, 219 suppressor, 9, 21 surgery, ix, 91, 92, 94, 95, 99, 103, 104, 105, 106, 107, 108, 117, 125, 128, 131, 133, 162, 165, 240 Surgery, 130, 178 surgical, 92, 121, 311 surgical intervention, 121 survival, ix, 33, 91, 112, 188, 207, 217, 224, 230, 272, 279, 287, 297, 305, 316, 318, 320 susceptibility, ix, 24, 79, 89, 91, 97, 116, 125, 133, 165, 169, 179, 195, 214, 306, 309 swallowing, 259 sweets, 49 switching, 13 sympathetic, 33, 35, 58, 172 sympathetic nervous system, 33, 172 symptomatic treatment, 255 symptoms, x, 114, 135, 139, 149, 261, 277, 283, 300 synapse, 263 synapses, 258, 262, 263, 301 synaptic plasticity, 263, 291 syndrome, x, xi, 16, 112, 117, 119, 135, 137, 140, 142, 147, 148, 149, 150, 159, 160, 161, 162, 164, 168, 169, 170, 171, 172, 173, 174, 175, 176, 178, 180, 185, 188, 189, 270, 278, 288, 305, 316, 318

Index synergistic, viii, 26, 50, 100, 121 synergistic effect, 50 synovial fluid, 19 synthesis, viii, 6, 13, 14, 25, 26, 28, 29, 30, 32, 49, 60, 62, 94, 96, 100, 101, 108, 113, 121, 126, 143, 145, 146, 170, 173, 176, 177, 187, 196, 199, 204, 205, 212, 224, 226, 227, 228, 230, 231, 232, 237, 239, 244, 245, 250, 264, 304, 309, 311, 315 systemic circulation, 122, 165 systems, vii, 1, 2, 9, 12, 16, 17, 24, 27, 36, 41, 43, 46, 54, 137, 141, 145, 190, 215, 226, 233, 246, 262, 306, 310 systolic blood pressure, 44, 47, 50, 59, 61, 74, 81

T T cell, 71, 72, 195, 209, 236 T cells, 72, 195 T lymphocyte, 177, 195 T lymphocytes, 177, 195 tachycardia, 99, 106, 108, 109 tacrolimus, 118, 119 tangles, 263 tannins, 48 targets, 95, 97, 141, 183, 234, 267, 292 tau, 279, 287, 288, 293 tau pathology, 293 T-cell, 72, 209, 315 TE, 83, 212, 253, 254, 294 tea, 282, 293, 308, 319, 320 Tea, 308, 321 temporal, 150, 262, 269 temporal lobe, 269 tensile, 28 tensile stress, 28 tension, 115, 121, 131, 139, 142, 152, 314, 317 terminals, 263, 277 ternary complex, 6 TF, 87, 88, 132, 227, 232 TGF, 123, 139, 148, 150, 238 Thai, 152, 212 thalamus, 321 therapeutic agents, 132, 241, 247 therapeutic approaches, 64, 126, 186, 271, 283, 319 therapeutic interventions, xii, xiii, 80, 224, 257, 283 therapeutic targets, 292 therapeutics, 284 therapy, xii, 43, 46, 47, 50, 54, 81, 87, 89, 97, 99, 104, 105, 114, 125, 126, 130, 132, 133, 144, 145, 146, 148, 156, 169, 173, 174, 188, 199, 204, 205,

Index 206, 215, 221, 224, 240, 241, 242, 244, 245, 246, 254, 255, 272, 281, 282, 288, 292, 300, 309, 310, 319, 320, 322 thiamin, 271, 309 thiamin deficiency, 271 Thiamine, 292, 309 thiazide, 119 thiazide diuretics, 119 thiobarbituric acid, 14, 42, 203, 234 thioredoxin, 55, 58, 203, 217, 234, 251 Thomson, 182 thoracic, 32, 33 threat, 235 threonine, 279 threshold, 44, 201 thrombin, 28, 32, 34, 35, 38, 75 thrombocytopenia, x, 135, 148 thrombolytic therapy, 103 thrombomodulin, 138 thrombosis, 33, 42, 64, 66, 72, 88 thrombotic, 73 thromboxane, 28, 35, 45, 266 thymine, 7 thyroid, 179 tight junction, 27, 116 time, 12, 19, 34, 47, 70, 73, 74, 75, 79, 92, 93, 94, 96, 112, 114, 124, 139, 146, 195, 199, 201, 202, 204, 235, 238, 260, 264, 272, 275, 276, 282, 311 timing, 145, 148, 307 tissue, x, xiii, 3, 4, 5, 10, 15, 17, 21, 22, 28, 29, 31, 33, 34, 36, 47, 55, 66, 67, 69, 71, 76, 93, 94, 95, 96, 97, 103, 104, 120, 121, 122, 124, 126, 131, 132, 135, 136, 142, 161, 162, 163, 165, 168, 169, 170, 171, 176, 181, 183, 185, 196, 199, 204, 207, 210, 220, 226, 233, 234, 235, 236, 237, 238, 240, 247, 251, 260, 264, 276, 278, 299, 301, 305, 306, 308, 309, 312, 315, 317 tissue plasminogen activator, 71 TJ, 22, 24, 52, 53, 57, 62, 88, 106, 129, 131, 132, 183, 213, 216, 289, 292 TLR, 209 TLR4, 209 T-lymphocytes, 67, 71 TM, xiii, 16, 61, 62, 105, 109, 138, 217, 221, 250, 254, 295, 299, 300, 301, 303, 304, 306, 307, 308, 311, 317 TNF, 69, 72, 75, 145, 155, 163, 164, 165, 182, 187, 195, 196, 203, 232, 233, 235, 236, 237, 243, 245, 252 TNF-alpha, 165, 182

355

TNF-α, 69, 72, 75, 163, 164, 195, 203, 232, 233, 235, 236, 237, 243, 245 tobacco, 198 tobacco smoke, 198 Tocopherol, 79, 108, 109, 156, 282, 297 tocopherols, 46, 79, 154, 166, 241, 274 tocotrienols, 79, 241 tolerance, 163, 253 toll-like, 71, 209 Toll-like, 71, 196 total cholesterol, 65, 200 total energy, 226 toxic, x, 2, 3, 4, 5, 10, 38, 42, 111, 114, 118, 120, 122, 130, 131, 133, 142, 167, 199, 211, 231, 233, 234, 263, 266, 301 toxic effect, 199, 263, 266 toxic products, 168 toxic substances, 114, 234 toxicity, xi, 4, 19, 22, 116, 125, 129, 193, 194, 199, 200, 203, 205, 207, 208, 209, 213, 214, 217, 231, 232, 241, 248, 250, 252, 264, 272, 281, 287, 302, 316 toxicology, 250 toxins, 72, 114, 117, 118, 165 trabeculae, 33 traits, 169, 277 trans, 231 transcription, 8, 85, 96, 97, 173, 200, 214, 227, 232, 235, 249, 265, 272, 280 transcription factor, 200, 214, 227, 249, 272, 280 transcription factors, 200, 227, 249 transcriptional, 13, 95, 96, 97, 100, 101, 104, 124, 132, 145 transduction, 182 transection, 314 transfer, 35, 39, 41, 76, 173, 183, 184, 218, 228, 229, 265, 267 transference, 140 transferrin, 153 transformation, 73, 234, 238 transforming growth factor, 93, 97, 139, 194, 238, 253 transgenic, 30, 52, 132, 168, 213, 217, 232, 251, 264, 266, 267, 271, 287, 289, 293 Transgenic, 184 transgenic mice, 30, 52, 213, 232, 266, 267, 271, 293 transgenic mouse, 217, 289, 293 transition, 4, 8, 225, 260, 264 transition metal, 4, 260, 264 translational, 13, 75

Index

356 translocation, 145, 155, 198, 270 transmembrane, 27, 263, 287, 315, 322 transmembrane glycoprotein, 263 transmission, 265 transplant, 124 transplantation, xii, 104, 121, 165, 223 transport, 7, 34, 41, 54, 76, 96, 115, 116, 117, 129, 130, 174, 179, 196, 198, 207, 210, 228, 232, 236, 258, 263, 264, 281, 310, 321, 322 transsulfuration, 167 trauma, 259 tremor, 277, 281 triacylglycerols, 236 trial, 50, 59, 60, 62, 79, 99, 103, 104, 108, 125, 144, 145, 154, 156, 173, 175, 183, 189, 190, 202, 203, 216, 220, 231, 242, 243, 244, 245, 247, 255, 269, 271, 273, 275, 276, 281, 282, 294, 297, 309, 310 triggers, 32, 116, 117, 156, 163, 164, 228, 230, 236, 263 triglyceride, 162, 173, 185, 201, 243 triglycerides, 68, 152, 224, 226, 245 tripeptide, 11, 265 trophoblast, x, 135, 137, 139, 141, 142, 144, 145, 146, 147, 148, 150, 153, 156, 157 trust, 280 tryptophan, 10 TT, 290 tubular, x, 111, 113, 114, 115, 116, 117, 118, 120, 121, 122, 127, 128, 129, 130, 131, 183, 279 tumor, 9, 21, 93, 97, 196, 202, 252, 290 tumor necrosis factor, 93, 97, 196, 252, 290 tumors, 9 tumour, 182, 215 turnover, 32, 95, 164, 187, 271, 272, 295 TXA2, 138 type 1 diabetes, 161, 183, 195, 198, 199, 201, 202, 203, 204, 209, 210, 212, 213, 216, 217, 220 type 2 diabetes, 12, 14, 23, 46, 48, 55, 60, 61, 161, 162, 170, 173, 179, 183, 184, 189, 191, 196, 199, 200, 202, 204, 208, 212, 215, 216, 219, 220, 221, 225, 250 type 2 diabetes mellitus, 23, 161, 162, 179, 184, 191, 202, 208, 212, 215, 219, 225 type II diabetes, 23, 178, 179, 210, 212, 233 tyrosine, 6, 9, 10, 21, 29, 35, 54, 154, 195, 234 tyrosyl radical, 6

U ubiquitin, 15

UK, 284 ulceration, 319 ultrasound, 155, 245, 319 ultraviolet, 48, 303, 308, 320 ultraviolet light, 320 UN, 113, 153, 188 uncoupling proteins, 281 underlying mechanisms, 97, 112, 172, 283 unfolded, 230, 250 unfolded protein response, 230, 250 uniform, 66 United States, xiii, 22, 216, 224, 257, 261, 275, 276, 284 unstable angina, 26, 166 UP, 83, 84 urban population, 186 urea, 112, 113, 114, 115, 126, 137 urethra, 114 uric acid, 5, 7, 11, 39, 87, 112, 118, 120, 137, 142, 144, 280, 296 uric acid levels, 137, 296 urinary, 107, 113, 114, 140, 144, 155, 173, 214 urinary tract, 114 urine, 17, 32, 37, 113, 114, 116, 118, 122, 136, 168, 280 uterus, 150 UV, 303, 316 UV radiation, 303

V Valencia, 62, 155 validation, 295 validity, 267 values, 14, 242 vancomycin, 117, 119 variables, 64, 143, 154, 164 variation, 80 vascular cell adhesion molecule, 85, 89, 212, 236 vascular disease, 22, 29, 37, 52, 54, 86, 88, 174, 178, 182, 194, 196, 200, 220, 291, 302 vascular diseases, 54, 174, 302 vascular endothelial growth factor, 75, 138, 147, 309 vascular endothelial growth factor (VEGF), 75 vascular inflammation, 42, 55, 212 vascular occlusion, 73 vascular reactions, 28 vascular risk factors, 174, 273 vascular surgery, 117 vascular system, 43, 234

Index vascular wall, viii, 5, 12, 26, 27, 28, 30, 36, 38, 44, 47, 50, 63, 64, 69, 72, 74, 80, 145 vascularization, 103, 104 vasculature, xi, 26, 28, 31, 33, 37, 38, 39, 42, 57, 70, 86, 137, 138, 193, 204, 237 vasculogenesis, 143, 147, 148 vasoconstriction, viii, 13, 25, 26, 28, 29, 30, 31, 37, 52, 70, 94, 115, 116, 118, 121, 122, 148 vasoconstrictor, 7, 26, 28, 31, 32, 33, 74, 201, 233 vasodilatation, 28, 36, 61, 62, 136, 145, 169, 171, 204, 205, 302 vasodilation, 13, 26, 27, 29, 31, 35, 37, 38, 41, 44, 51, 56, 70, 77, 79, 120, 155, 168, 173, 183, 189, 234, 303, 309, 321 vasodilator, viii, 25, 26, 29, 32, 33, 35, 36, 37, 41, 46, 53, 59, 71, 89, 101, 118, 264 vasomotor, viii, 25, 26, 27, 35, 83, 85, 115, 140 vasospasm, 15, 16, 29 VC, 289 VCAM, 69, 71, 72, 73, 85, 236 vegetables, 11, 49, 50, 79, 124, 173 VEGF, 75, 138, 147, 148 vein, 19, 89, 105, 115, 131, 307 velocity, 206 venous pressure, 301 ventricles, 93 ventricular fibrillation, 105 ventricular tachycardia, 106 vessels, 28, 29, 32, 61, 65, 70, 71, 200, 264 veterinarians, 170 victims, 261, 268, 270 VIP, 156 viral hepatitis, 235 virus, 8, 162 visceral adiposity, 162, 164, 170, 179 viscosity, 109 vision, 311 visual field, 300, 304, 312 vitamin A, 273 vitamin B1, 206, 269, 271, 291 vitamin B12, 269, 271, 291 vitamin B6, 206, 269, 291 vitamin C deficiency, 124 vitamin E, viii, 11, 24, 25, 41, 42, 43, 45, 48, 50, 59, 60, 78, 79, 89, 90, 92, 99, 100, 101, 102, 108, 125, 126, 133, 143, 145, 154, 156, 166, 171, 172, 174, 187, 188, 190, 202, 203, 216, 217, 239, 241, 242, 244, 247, 253, 255, 267, 271, 272, 273, 274, 275, 276, 290, 293, 294, 303, 309, 310, 311, 315, 322

357

vitamin supplementation, 242, 282, 307, 310 vitamins, viii, x, xi, 17, 25, 46, 47, 58, 60, 99, 100, 101, 102, 103, 104, 108, 109, 125, 136, 141, 144, 145, 146, 147, 148, 155, 156, 159, 166, 170, 171, 172, 183, 187, 189, 201, 204, 216, 218, 241, 273, 275, 285, 291, 294, 321 vitreous, 302, 303, 314, 315 VLDL, 73, 162, 226 VO, 180, 184, 248 vulnerability, 104, 126, 165, 306, 318

W waste products, 112 wastes, 115 water, ix, 3, 11, 32, 41, 44, 90, 100, 111, 112, 113, 117, 118, 231, 244, 311, 312 water-soluble, 11, 44, 100, 244, 311 wavelets, 93, 94 web, 97 weight gain, 178 weight loss, 174, 176, 179, 225, 239, 240, 242 weight reduction, 252 Weinberg, 130 Western countries, 224 WG, 105, 247, 291 wheat, 174 wheat germ, 174 white blood cell count, 107 white blood cells, 107 white matter, 291 WHO, 22 wild type, 272, 279 wine, 48, 50, 62, 126, 134, 309, 320, 321 Wisconsin, 184 Wistar rats, 185 WM, 62, 88, 129, 188, 220, 253, 289, 290, 297, 315, 319 Wnt signaling, 288 women, x, 31, 44, 135, 136, 140, 141, 142, 143, 144, 145, 146, 148, 152, 153, 154, 155, 156, 171, 182, 187, 189, 190, 216, 274, 276, 293, 294 World Health Organization, 81 wound healing, 252 WP, 81, 87, 180, 215, 296, 297

X xenobiotic, 231, 233

Index

358 xenobiotics, 231 xenografts, 321

Y yang, 320 yeast, 21 yield, 39 yin, 320 yogurt, 49 young adults, 203

Z zebrafish, 313 zinc, 187, 195, 202, 273, 275, 294 Zn, 124, 203, 217, 265