Aging oxidative stress and dietary antioxidants [2nd ed] 9780128186985, 1501511521, 2012022022, 2672682712, 2332332332, 3193213233, 3273273283, 0128186984, 9780128188118, 0128188111

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AGING

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AGING

Oxidative Stress and Dietary Antioxidants SECOND EDITION Edited by

VICTOR R. PREEDY VINOOD B. PATEL

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-818698-5 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Mary Preap Editorial Project Manager: Timothy Bennett Production Project Manager: Swapna Srinivasan Cover Designer: Greg Harris Typeset by SPi Global, India

Contents Contributors Preface

Recent advances in cardiovascular disease, aging and oxidative stress Applications to other diseases and conditions Summary points References Further reading

xi xv

1 Oxidative stress and aging

4. Aging, oxidative stress, mitochondrial dysfunction, and the liver

1. Oxidative stress and miR-200c

JANICE G. LOZADA-DELGADO, CARLOS A. TORRES-RAMOS, ˜A SYLVETTE AYALA-PEN

ALESSANDRA MAGENTA, MARIA CRISTINA FLORIO, MARCO D’AGOSTINO, SARA SILENO

Introduction miR-200c and oxidative stress Applications to other diseases or conditions Summary points References

Introduction Evidence for age-associated morphological, structural, and functional changes in the liver Evidence for age-associated loss of mitochondrial bioenergetics in liver Oxidative stress and antioxidant responses in liver aging Oxidative damage to the nuclear and mitochondrial genomes in liver aging Mitochondrial dysfunction and liver-associated disease Conclusion Summary points References

3 5 8 9 9

2. Caloric restriction, reactive oxygen species, and longevity MIYUKI KOBARA, HIROE TOBA, TETSUO NAKATA

Introduction CR in animals Mechanisms responsible for beneficial effects of CR in animals CR in humans Trials on other modified CR and other modules mimicking CR-induced beneficial signaling Conclusions and future prospective questions Applications to other diseases or conditions Summary points References Further reading

11 11

37 38 38 39 40 42 44 44 45

12 13

5. Culprit effect of oxidative stress in the development of Parkinson’s disease: A conundrum of senescence

15 16 16 16 16 18

RAHUL SAXENA, GLADYS RAI

Introduction Aging and Parkinson disease: A mysterious convergence Treacherous role of oxygen and nitrogen molecule in PD Neuroinflammation in PD Therapeutic interventions Summary points References Further reading

3. Cardiovascular disease in aging and the role of oxidative stress LUCIE ORLIAGUET, VINOOD B. PATEL, VICTOR R. PREEDY, FAWAZ ALZAID

Introduction Physiology of the aging cardiovascular system The molecular basis of oxidative stress as applied to the cardiovascular system Longevity genes and the longevity network Genes of the longevity network: SIR2, IGF-1, AMPK, and mTOR The longevity network: SIR2, IGF1, AMPK, and mTOR Other genes and pathways in oxidative stress and age-related cardiovascular disease

30 31 32 32 35

47 48 48 56 57 57 58 59

6. Linking bone cells, aging, and oxidative stress: Osteoblasts, osteoclasts, osteocytes, and bone marrow cells

19 21

´ LVAREZ-CARRIO ´ N, JUAN A. ARDURA, LUIS A ´ ZAR, VERO ´ NICA ALONSO ARANCHA R. GORTA

23 24

Basic biology of bone Aging and bone: Role of oxidative stress Molecular mechanisms that mediate oxidative stress in aging bone cells Summary points References

24 27 27

v

61 62 63 69 69

vi

Contents

7. Oxidative stress, senescence and Mediterranean diet effects on osteoarthritis SERGIO AMMENDOLA, ANNA SCOTTO D’ABUSCO

Introduction Chondrocytes in osteoarthritis Chondrocytes and senescence Chondrocytes and oxidative stress Chondrocytes and pro- and antioxidant agents Mediterranean nutrients: Protective or risk factors? Conclusions Application to other diseases Summary points References

73 74 74 76 77 78 79 79 80 80

83 84 92 92

9. Oxidative stress and sarcopenia FRANCESCO BELLANTI, AURELIO LO BUGLIO, GIANLUIGI VENDEMIALE

95 95 97 98 99 100 101 101

10. Oxidative stress and hypertension in old age: The role of physical exercise

Introduction Menopausal symptoms Oxidative stress, blood antioxidant status, and menopausal symptoms Antioxidant-rich diets and menopausal symptoms Antioxidants supplementation in menopausal symptoms management Antioxidants and minerals Summary points References

126 127 128 129 131 131

2 Antioxidants and aging 13. Reference dietary requirements of antioxidant vitamins in the older adults

105 106 106 109 109 109 109

Introduction Oxidative stress Reference dietary requirements Vitamin A Vitamin C (ascorbic acid) Vitamin E Other vitamins Challenges in meeting reference dietary requirements in older adults Applications to other diseases or conditions Summary points References

11. Linking senescence, apoptosis, and oxidative stress in fertility

14. Oxidative stress and antioxidants in elderly women

MATHIAS ABIODUN EMOKPAE, OSARETIN GODWIN IGHARO

BRUNNA CRISTINA BREMER BOAVENTURA, PATRICIA FARIA DI PIETRO, FRANCIELI CEMBRANEL

Introduction Changing maternal age in some countries Senescence enhances free radical generation

125 125

MINA YAMAZAKI PRICE, VICTOR R. PREEDY

´ , VANESSA CORRALO, ˆ NIO DE SA CLODOALDO ANTO FRANCIELLE GARGHETTI BATTISTON, MARIA ISABEL GONC ¸ ALVES DA SILVA

Introduction Aging, oxidative stress, and hypertension Physical exercise, oxidative stress, and blood pressure control Conclusion Applications to other diseases or conditions Summary points References

117 118 119 119 120 120 120 121 122 122

GITY SOTOUDEH, MARYAM ABSHIRINI

RAHUL SAXENA, JYOTI BATRA

Introduction The aging muscle and sarcopenia Reactive species and redox signaling in aging Redox signaling in skeletal muscle Redox alterations and sarcopenia Applications to other diseases or conditions Summary points References

115 115 117

12. Antioxidant capacity and menopausal symptoms

8. Arthritis as a disease of aging and changes in antioxidant status Introduction The concept of oxygen toxicity and free radicals Summary References

Free radical theory of aging Involvement of other biomolecules in senescence Mechanisms of senescence in reproduction Evidence linking senescence, apoptosis, and oxidative stress in fertility Senescence exacerbates apoptosis Senescence alters endocrine system Senescence alters sex hormone levels The impact of senescence on sexual organs Impact of senescence on female reproductive function Senescence and ovarian function Applications to other diseases Summary points References

113 114 114

Introduction Review

137 137 138 138 138 138 139 143 143 143 144

145 145

Contents

Role of exercise on oxidative stress and antioxidant status of elderly women Applications to other diseases or conditions Summary points References

150 151 152 152

15. Caffeine and its analogs, antioxidants and applications 155 155 156 157 158 159 160 162 162 162 162

ELENA M. YUBERO-SERRANO, FRANCISCO M. GUTIERREZ-MARISCAL, ANTONIO GARCIA-RIOS,  JAVIER DELGADO-LISTA, PABLO PEREZ-MARTINEZ, ANTONIO CAMARGO, FRANCISCO PEREZ-JIMENEZ, JOSE LOPEZ-MIRANDA

165 165 166 166 167 167 168 168 169 170 170

17. The role of coenzyme Q10 in the protection of bone health during aging

Introduction Bioenergetics and antioxidant activity of CoQ10 CoQ10 and longevity, the intriguing role of Clk-1/COQ7 CoQ10 levels and longevity in humans CoQ10 in age-related diseases CoQ1010 supplementation and longevity Applications to other diseases or conditions Summary points References

183 184 184 186 187 187 188 189 189

19. Crocus sativus L. (saffron) extract antioxidant potential and use in aging SAEED SAMARGHANDIAN, TAHEREH FARKHONDEH, TAYEBEH ZEINALI

Background Antioxidant system Mode of action of saffron Chemical composition of saffron Safety assessments of saffron and its main ingredients Saffron and its main ingredients, oxidative stress, and aging References

193 194 194 194 195 196 199

20. Pharmacological profile of γ-oryzanol: Its antioxidant mechanisms and its effects in age-related diseases WIRAMON RUNGRATANAWANICH, GIULIA ABATE, DANIELA UBERTI

Introduction Pharmacokinetics of γ-oryzanol Pharmacodynamics of γ-oryzanol Antioxidant effects of γ-oryzanol in age-related diseases Summary points References

201 202 202 204 206 206

21. Herbs including shell ginger, antioxidant profiles, aging, and longevity in Okinawa, Japan: A critical analysis of current concepts

´ RQUEZ, MARI´A D. NAVARRO L. QUILES, JOSE  M. ROMERO-MA JOSE ´ PEZ HORTAL, MAURIZIO BATTINO, ALFONSO VARELA-LO

Bone biology Regulation of bone remodeling Reactive oxygen species (ROS) in bone remodeling Oxidative stress and bone health Aging at the bone Coenzyme Q10 (CoQ10), oxidative stress, and bone metabolism

180 181 181

´ CIDO NAVAS ´ PEZ-LLUCH, PLA GUILLERMO LO

16. Coenzyme Q10 as an antioxidant in the elderly

Aging Coenzyme Q10 Coenzyme Q10 functions Levels and distribution of coenzyme Q10 in the human organism Biosynthesis and transport of CoQ10 Uptake and distribution of CoQ10 CoQ10 and aging Therapeutic uses of CoQ10 in age-related diseases Conclusions Summary points References

178

18. Coenzyme Q10 supplementation in aging

BEATA JASIEWICZ, ARLETA SIERAKOWSKA

Introduction Oxidative stress and reactive oxygen species Antioxidant properties of caffeine and its analogs Caffeine and its analogs as adenosine receptors antagonists Caffeine and its analogs as monoamine oxidase inhibitors Caffeine and its analogs in neurodegenerative disease Anticancer properties of caffeine and its analogs Antibacterial and antifungal activity of caffeine analogs Caffeine and its analogs in medicines, cosmetics, and dietary supplements Summary points References

What happens during aging? Has diet any modulatory role in CoQ10 effect on bone health during aging? Summary points References

vii

173 174 175 175 176 176

ROLF TESCHKE, TRAN DANG XUAN

Introduction Literature search Definitions Specificities of plant use Shell ginger and selected plants with their antioxidant profiles and biological activities

209 210 210 211 212

viii Issue of plant antioxidants in aging or longevity and its evidence Okinawa plants, their antioxidants and association with longevity Longevity-specific bioactivities of A. zerumbet Okinawa longevity Causality gap under discussion between antioxidant plants and longevity in Okinawa Conclusions Summary points References

Contents

214 215 216 217 219 220 220 221

22. Lycopene as an antioxidant in the prevention and treatment of postmenopausal osteoporosis 223 223 224 225 229 229 230

259 259 260 260 261 263 263 264

Introduction Natural products in aging skin Discussion Conclusion Summary points References

267 268 271 272 272 272

27. Antioxidant properties and applications of Ophiopogon japonicus root for age-related disease YUMI KITAHIRO, MAKIO SHIBANO

WASEEM HASSAN, MEHREEN ZAFAR, JEAN PAUL KAMDEM

233 233 233 235 237 238 240 240

24. Antioxidant and antiinflammatory role of melatonin in Alzheimer’s neurodegeneration

Introduction Features of cellular senescence Ophiopogonis Radix Antiinflammatory effects of Ophiopogonis Radix on senescent NHDFS Kampo, the traditional Japanese medicine Conclusions Summary points References

275 275 276 277 279 279 281 282

28. Passion fruit seed: Its antioxidative extracts and potency in protection of skin aging NATTAYA LOURITH, MAYUREE KANLAYAVATTANAKUL

SERGIO A. ROSALES-CORRAL, RUSSEL J. REITER, XIAOYAN LIU

Introduction How free radicals are formed Some oxidative stress-related facts about the brain Where do free radicals come from in the Alzheimer’s disease brain? How does melatonin scavenge free radicals? Melatonin stimulates antioxidant systems Breaking the cycle neuroinflammation $ oxidative stress Melatonin production decreases with age Conclusions Applications to other diseases or conditions Summary points References

Introduction Mechanisms of free radical production Antioxidant defense system Oxidative stress and aging Murici [Byrsonima crassifolia (L.) Kunth] Conclusion Summary points References

MAHENDRAN SEKAR

23. Medicinal plants, antioxidant potential, and applications to aging Introduction The free radical theory of aging Free radical-induced damages Antiaging effects of medicinal plants Some of the major antioxidant enzymes and nonenzymes of medicinal plants and their proposed mechanism of action Some important points to be noted Summary points References

 MARIANA SEFORA BEZERRA SOUSA, DIEGO DE SOUZA BUARQUE

26. Natural products in aging skin

L.G. RAO, E.S. MACKINNON, A.V. RAO

Introduction Oxidative stress and antioxidants Osteoporosis Studies on lycopene Summary points Acknowledgments References

25. Murici (Byrsonima crassifolia (L.) Kunth): Antioxidant effects and application to aging

243 244 246 247 250 252 252 253 253 254 254 254

Introduction Oxidative stress and skin aging Antioxidative passion fruit seed extract and its potency in skin protection Conclusion Applications to other diseases or conditions Summary points References

283 283 284 285 287 287 287

29. Phaseolus vulgaris L. as a functional food for aging protection EUNICE SANTOS, GUILHERMINA MARQUES, TERESA LINO-NETO

Introduction Nutritional importance of common bean Occurrence of bioactive compounds in common bean

289 289 290

Contents

Polyphenols as important antioxidant compounds in common bean Other antioxidants in common bean Common bean and prevention of aging processes Conclusions Applications to other diseases or conditions Summary points Acknowledgments References

291 292 292 293 294 294 294 294

30. Quercetin and resveratrol, aging and kidney MINA HEMMATI, MARYAM MOOSSAVI

Introduction Diabetes and renal cell aging Transforming growth factor beta (TGF-β) Tumor necrosis factor alpha (TNF-α) Advanced glycation end product (AGE) NADPH oxidase Endothelin-1 (ET-1) Urotensin II Cannabinoid receptor (CB) Beta-galactosidase as a marker of senescent cells Summary points References

297 298 299 299 299 299 299 300 300 300 301 301

31. Rambutan fruits extract in aging skin MAHENDRAN SEKAR

Introduction Minerals in rambutan peel Polyphenolic compounds in rambutan peel Role of polyphenolic compounds in antiaging Relationship of antioxidants with antiaging therapy Antioxidant activity of various parts of rambutan fruits Antiaging effect of rambutan fruits Conclusion Summary points References

303 303 304 304

33. Resveratrol, SIRT1, oxidative stress, and brain aging FIORELLA SARUBBO, SILVIA TEJADA, SUSANA ESTEBAN, MANUEL JIMENEZ-GARCI´A, DAVID MORANTA

Brain aging: Role of oxidative stress and sirtuins Resveratrol: Their effect in brain aging through SIRT1 Challenges on resveratrol application in the prevention of neurodegeneration Applications to other diseases or conditions Summary points Acknowledgments References

319 321 323 324 324 324 324

34. Antioxidants, vegetarian diets, and age-related disease ANDREW C. BULMER, SHAILENDRA ANOOPKUMAR-DUKIE, IRIS F.F. BENZIE

Introduction Human aging and its consequences Oxidative stress and age-related disease Potential role of vegetarian diets for lowering risk of disease and increasing health span Some evidence Questions of molecular connections and research needs Summary points References

327 327 328 330 331 333 334 334

35. Protective effect of vitamin D on oxidative stress in elderly people

304

BRUNNA CRISTINA BREMER BOAVENTURA, FRANCIELI CEMBRANEL

304 305 305 306 306

Introduction Review Applications to other diseases or conditions Summary points References

337 337 342 342 342

36. Zinc, oxidative stress in the elderly, and implication for inflammation

32. Resveratrol, aging, and fatigue LUANA TONIOLO, EMILIANA GIACOMELLO

Introduction Skeletal muscle fatigue ROS and muscle fatigue Sarcopenia and lifestyle Calorie restriction and aging Resveratrol is a potent antioxidant with multiple functions Resveratrol and exercise in animal models Resveratrol and exercise in humans Resveratrol and fatigue, the underlying mechanisms Conclusions Summary points References

ix

309 309 310 311 311 311 312 313 313 315 315 315

ANANDA S. PRASAD, BIN BAO

Introduction Discovery of zinc deficiency in humans Clinical manifestations of zinc deficiency Zinc deficiency in the elderly subjects Effect of zinc supplementation in the elderly: Cell-mediated immunity and incidence of infection Effect of zinc supplementation on oxidative stress and inflammatory cytokines in the elderly Zinc trials in AMD Zinc and NF-κB activation Proposed concept of mechanism of zinc action as an antioxidant and antiinflammatory agent

345 345 346 347 348 352 354 355 355

x Mechanism of zinc action as an antioxidant Zinc as a molecular signal regulating oxidative stress and chronic inflammation Metallothionein Summary points References

Contents

356 357 358 359 359

37. Oxidative stress and the senescence acceleration in senescence-accelerated mouse P10 (SAMP10) TAKESHI SAITO, MASAAKI KURASAKI

Free radical theory of aging Senescence-accelerated mouse: Animal model of aging Oxidative stress and aging of SAMP10 Production of reactive oxygen species (ROS) in SAMP10 Production of ROS in mitochondria of SAMP10 mice with aging Production of neuronal NO by NO synthase

363 363 364 364 364 364

Production of oxidative stress in glutamatergic neurotransmission Changes in antioxidant defense systems of SAM10 in aging Conclusion Summary points References

365 365 366 366 366

38. Recommended resources on aging: Oxidative stress and dietary antioxidants RAJKUMAR RAJENDRAM, VINOOD B. PATEL, VICTOR R. PREEDY

Introduction Resources Summary points Acknowledgments References

Index

369 370 372 373 373

375

Contributors

Giulia Abate Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

Aurelio Lo Buglio Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy

Maryam Abshirini School of Health Sciences, College of Health, Massey University, Palmerston North, New Zealand

Andrew C. Bulmer School of Medical Science, Griffith University, Gold Coast, QLD, Australia

Verónica Alonso Department of Basic Medical Sciences, School of Medicine, San Pablo CEU University, CEU Universities, Alcorcón, Madrid, Spain

Antonio Camargo Lipid and Atherosclerosis Unit, IMIBIC/ Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain

Luis Álvarez-Carrión Bone Physiopathology Laboratory, Applied Molecular Medicine Institute (IMMA), Universidad San Pablo-CEU, CEU Universities, Alcorcón, Madrid, Spain

Francieli Cembranel Department of Nutrition, Health Sciences Center, Federal University of Santa Catarina, Florianópolis, SC, Brazil

Fawaz Alzaid Immunity and Metabolism of Diabetes Team, INSERM Unit 1138, Cordeliers Research Centre, Paris, France

Vanessa Corralo Postgraduate Program on Health Sciences, Unochapecó University, Chapecó, Santa Catarina, Brazil

Sergio Ammendola Ambiotec S.A.S., Cisterna di Latina, Italy

Marco D’Agostino Experimental Immunology Laboratory, Dermopatic Institute of Immaculate-IDIIRCCS, Rome, Italy

Shailendra Anoopkumar-Dukie School of Pharmacy & Pharmacology, Griffith University, Gold Coast, QLD, Australia Juan A. Ardura Department of Basic Medical Sciences, School of Medicine, San Pablo CEU University, CEU Universities, Alcorcón, Madrid, Spain

Javier Delgado-Lista Lipid and Atherosclerosis Unit, IMIBIC/ Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain

Sylvette Ayala-Peña Department of Pharmacology and Toxicology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, United States

Patricia Faria Di Pietro Department of Nutrition, Health Sciences Center, Federal University of Santa Catarina, Florianópolis, SC, Brazil

Bin Bao Department of Oncology, Wayne State University School of Medicine, and Barbara Ann Karmanos Cancer Institute, Detroit, MI, United States

Mathias Abiodun Emokpae Department of Medical Laboratory Science, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Nigeria

Jyoti Batra Dean Research & Professor, Department of Biochemistry, Santosh Medical College and Hospital, Santosh University, Ghaziabad, Uttar Pradesh, India

Susana Esteban Neurophysiology Group, Biology Department, University of Balearic Islands, Palma, Balearic Islands, Spain

Maurizio Battino Nutrition and Food Science Group, Department of Analytical and Food Chemistry, CITACA, CACTI, University of Vigo, Vigo, Spain; Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche (DISCO)-Sez. Biochimica, Facoltà di Medicina, Università Politecnica delle Marche, Ancona, Italy

Tahereh Farkhondeh Cardiovascular Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran

Francielle Garghetti Battiston Postgraduate Program on Health Sciences, Unochapecó University, Chapecó, Santa Catarina, Brazil Francesco Bellanti Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy

Antonio Garcia-Rios Lipid and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain

Iris F.F. Benzie School of Pharmacy & Pharmacology, Griffith University, Gold Coast, QLD, Australia

Emiliana Giacomello Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy

Maria Cristina Florio Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Biomedical Research Center, Baltimore, MD, United States

Maria Isabel Gonc¸ alves da Silva Postgraduate Program on Health Sciences, Unochapecó University, Chapecó, Santa Catarina, Brazil

Brunna Cristina Bremer Boaventura Department of Nutrition, Health Sciences Center, Federal University of Santa Catarina, Florianópolis, SC, Brazil

xi

xii

Contributors

Arancha R. Gortázar Department of Basic Medical Sciences, School of Medicine, San Pablo CEU University, CEU Universities, Alcorcón, Madrid, Spain Francisco M. Gutierrez-Mariscal Lipid and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain Waseem Hassan Institute of Chemical Sciences, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan Mina Hemmati Biochemistry Department, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Osaretin Godwin Igharo Department of Medical Laboratory Science, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Nigeria Beata Jasiewicz Faculty of Chemistry, Adam Mickiewicz University in Pozna n, Uniwersytetu Pozna nskiego, Pozna n, Poland Manuel Jimenez-García Neurophysiology Group, Biology Department, University of Balearic Islands, Palma, Balearic Islands, Spain Jean Paul Kamdem Department of Biological Sciences, Regional University of Cariri, Crato, Ceara, Brazil Mayuree Kanlayavattanakul School of Cosmetic Science; Phytocosmetics and Cosmeceuticals Research Group, Mae Fah Luang University, Chiang Rai, Thailand Yumi Kitahiro Department of Natural Products Research, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan Miyuki Kobara Department of Clinical Pharmacology, Division of Pathological Science, Kyoto Pharmaceutical University, Kyoto, Japan Masaaki Kurasaki Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Japan Teresa Lino-Neto Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), University of Minho, Braga, Portugal Xiaoyan Liu School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China Guillermo López-Lluch Centro Andaluz de Biología del Desarrollo (CABD-CSIC-JA) (CIBERER), Instituto de Salud Carlos III, Universidad Pablo de Olavide, Sevilla, Spain Jose Lopez-Miranda Lipid and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain

E.S. Mackinnon Department of Medicine, St. Michael’s Hospital; University of Toronto, Toronto, ON, Canada Alessandra Magenta Experimental Immunology Laboratory, Dermopatic Institute of Immaculate-IDI-IRCCS, Rome, Italy Guilhermina Marques Centre for Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal Maryam Moossavi Student Research Committee, Birjand University of Medical Sciences, Birjand, Iran David Moranta Neurophysiology Group, Biology Department, University of Balearic Islands, Palma, Balearic Islands, Spain Tetsuo Nakata Department of Clinical Pharmacology, Division of Pathological Science, Kyoto Pharmaceutical University, Kyoto, Japan María D. Navarro-Hortal Institute of Nutrition and Food Technology “Jose Mataix Verdú,” Department of Physiology, Biomedical Research Center, University of Granada, Granada, Spain Plácido Navas Centro Andaluz de Biología del Desarrollo (CABD-CSIC-JA) (CIBERER), Instituto de Salud Carlos III, Universidad Pablo de Olavide, Sevilla, Spain Lucie Orliaguet Immunity and Metabolism of Diabetes Team, INSERM Unit 1138, Cordeliers Research Centre, Paris, France Vinood B. Patel Department of Biomedical Sciences, School of Life Sciences, University of Westminster, London, United Kingdom Francisco Perez-Jimenez Lipid and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain Pablo P erez-Martinez Lipid and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain Ananda S. Prasad Department of Oncology, Wayne State University School of Medicine, and Barbara Ann Karmanos Cancer Institute, Detroit, MI, United States Victor R. Preedy Diabetes and Nutritional Sciences Research Division, School of Medicine; Department of Nutritional Sciences, School of Life Course Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom

Nattaya Lourith School of Cosmetic Science; Phytocosmetics and Cosmeceuticals Research Group, Mae Fah Luang University, Chiang Rai, Thailand

Mina Yamazaki Price Division of Critical Care, Medicine and Surgery, Department of Therapies, Royal Free Hospital, Royal Free London NHS Foundation Trust, London, United Kingdom

Janice G. Lozada-Delgado Department of Pharmacology and Toxicology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, United States

Jos e L. Quiles Institute of Nutrition and Food Technology “Jose Mataix Verdú,” Department of Physiology, Biomedical Research Center, University of Granada, Granada, Spain

Contributors

xiii

Gladys Rai Department of Biochemistry, School of Medical Sciences & Research, Sharda University, Greater Noida, Uttar Pradesh, India

Arleta Sierakowska Faculty of Chemistry, Adam Mickiewicz University in Pozna n, Uniwersytetu Pozna nskiego, Pozna n, Poland

Rajkumar Rajendram College of Medicine, King Saud bin Abdulaziz University for Health Sciences; Department of Medicine, King Abdulaziz Medical City, Riyadh, Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia; Diabetes and Nutritional Sciences Research Division, Faculty of Life Science and Medicine, King’s College London, London, United Kingdom

Sara Sileno Experimental Immunology Laboratory, Dermopatic Institute of Immaculate-IDI-IRCCS, Rome, Italy

A.V. Rao Department of Nutritional Sciences, University of Toronto, Toronto, ON, Canada L.G. Rao Department of Medicine, St. Michael’s Hospital; University of Toronto, Toronto, ON, Canada Russel J. Reiter Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States Jose M. Romero-Márquez Institute of Nutrition and Food Technology “Jose Mataix Verdú,” Department of Physiology, Biomedical Research Center, University of Granada, Granada, Spain Sergio A. Rosales-Corral Western Biomedical Research Center from the Mexican Institute of Social Security, Guadalajara, Jalisco, Mexico Wiramon Rungratanawanich Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy Clodoaldo Antônio De Sá Postgraduate Program on Health Sciences, Unochapecó University, Chapecó, Santa Catarina, Brazil Takeshi Saito Faculty of Health Sciences, Hokkaido University, Sapporo, Japan Saeed Samarghandian Healthy Ageing Research Center, Neyshabur University of Medical Sciences, Neyshabur, Iran Eunice Santos Centre for Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real; Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), University of Minho, Braga, Portugal

Gity Sotoudeh Department of Community Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran Mariana S efora Bezerra Sousa Nutrition Department, Federal Institute of Ceará (IFCE), Iguatu, Brazil Diego de Souza Buarque Academic Unit of Serra Talhada (UAST), Rural Federal University of Pernambuco (UFRPE), Serra Talhada, Brazil Silvia Tejada Neurophysiology Group, Biology Department; Biology Department and CIBEROBN (Physiopathology of Obesity and Nutrition), University of Balearic Islands, Palma, Balearic Islands, Spain Rolf Teschke Department of Internal Medicine, Division of Gastroenterology and Hepatology, Klinikum Hanau, Hanau; Academic Teaching Hospital of the Medical Faculty, Goethe University Frankfurt/Main, Frankfurt/Main, Germany Hiroe Toba Department of Clinical Pharmacology, Division of Pathological Science, Kyoto Pharmaceutical University, Kyoto, Japan Luana Toniolo Department of Biomedical Sciences, University of Padova, Padova, Italy Carlos A. Torres-Ramos Department of Physiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, United States Daniela Uberti Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy Alfonso Varela-López Institute of Nutrition and Food Technology “Jose Mataix Verdú,” Department of Physiology, Biomedical Research Center, University of Granada, Granada, Spain Gianluigi Vendemiale Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy

Fiorella Sarubbo Neurophysiology Group, Biology Department, University of Balearic Islands; Research Unit, Hospital Universitario Son Llàtzer, Health Research Institute of Balearic Islands (IdISBa), Palma, Balearic Islands, Spain

Tran Dang Xuan Division of Development Technology, Graduate School for International Development and Cooperation (IDEC), Hiroshima University, Higashi Hiroshima, Japan

Rahul Saxena Department of Biochemistry, School of Allied Health Sciences, Sharda University, Greater Noida, Uttar Pradesh, India

Elena M. Yubero-Serrano Lipid and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba, Córdoba; CIBER Fisiopatologia Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain

Anna Scotto d’Abusco Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy Mahendran Sekar Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Health Sciences, Universiti Kuala Lumpur, Royal College of Medicine Perak, Ipoh, Malaysia Makio Shibano Department of Natural Products Research, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan

Mehreen Zafar Institute of Chemical Sciences, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan Tayebeh Zeinali Social Determinants of Health Research Center, School of Health, Birjand University of Medical Sciences, Birjand, Iran

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Preface

in Section 1, there is coverage of oxidative stress and aging in cardiovascular disease, arthritis, the liver, fertility, sarcopenia, and calorie restriction. In Section 2 the focus is on antioxidants, covering recommended amounts in the elderly, Coenzyme Q10 supplementation, antioxidants and vegetarian diets, and caffeine analogues; natural antioxidants such as lycopene, zinc, and ginger; and plant-derived products including, Murici extract, Ophiopogonis radix root, passion fruit seed, and saffron extract, where models of aging are also discussed. Thus this text is relevant to gerontologist, geriatricians, nutritionists, dieticians, and nutrition researchers as aging is a multifaceted process covering disease processes, clinical research, and treatment.

Here is this book Aging: Oxidative Stress and Dietary Antioxidants, Second Edition that bridges the transdisciplinary divide and covers the science of oxidative stress in aging and the therapeutic use of natural antioxidants in the food matrix in a single volume. The second edition covers new trials and investigations used to determine the comprehensive properties of antioxidants, food items and extracts, and any adverse properties they may have. It has been updated to include new clinical human trials and studies dedicated to animal models of aging. Throughout the book the processes within the science of oxidative stress are described in concert with other processes, such as apoptosis, cell signaling, and receptor-mediated responses. This approach recognizes that diseases are often multifactorial and oxidative stress is a single component of this. Here is this book Aging: Oxidative Stress and Dietary Antioxidants, Second Edition that contains two sections:

Victor R. Preedy and Vinood B. Patel

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S E C T I O N

1

Oxidative stress and aging

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C H A P T E R

1 Oxidative stress and miR-200c Alessandra Magentaa, Maria Cristina Floriob, Marco D’Agostinoa, Sara Silenoa a

b

Experimental Immunology Laboratory, Dermopatic Institute of Immaculate-IDI-IRCCS, Rome, Italy Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Biomedical Research Center, Baltimore, MD, United States

enzymes such as several oxidases, peroxidase, cytochromes, mono- and dioxygenases, and uncoupled nitric oxide synthase (NOS).1 Enzymatic and nonenzymatic antioxidant defenses such as superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione finely modulate the amount of ROS within the cell.2 Insufficient scavenging or ROS production exacerbation has been demonstrated to impair many biological processes. There is a close link between NOS activity and ROS production, since uncoupling of NOS leads to the production of superoxide anion, rather than NO.3,4 The cellular transduction pathways induced by the increase in ROS are known to cause growth arrest and senescence, as well as cell death, both by apoptosis and necrosis, based on the level of oxidative stress experienced by the cell and its genotype. A mechanism of induction of apoptosis from oxidative stress involves the p53 tumor suppressor protein; p53, in turn, regulates the intracellular redox state and induces apoptosis through a mechanism involving ROS production.5 A key role in ROS-induced apoptosis is also played by the p66 isoform of the ShcA protein (p66Shc), a fundamental regulator of mitochondrial ROS production from a variety of different stimuli.6 Different studies underlined the role of microRNAs (miRNAs) in cellular homeostasis and in the regulation of redox balance.

List of abbreviations BCNU CAT ECs EMT eNOS FOXO1 GPx HEI-OC1 H2O2 MAPK miRNA MnSOD mRNA NAC NOS NOX p66Shc ROS pRb RISC SIRT1 SOD TRBP t-BHP Xpo-5 ZEB1

1,3-bis(2 chloroethyl)-1-nitrosourea catalase endothelial cells epithelial-mesenchymal transition endothelial nitric oxide synthase forkhead box O1 glutathione peroxidase House Ear Institute-Organ of Corti 1 hydrogen peroxide mitogen-activated protein kinase microRNA manganese superoxide dismutase messenger RNA N-acetyl-L-cysteine nitric oxide synthase NADPH oxidase p66 isoform of ShcA protein reactive oxygen species retinoblastoma protein RNA-induced silencing complex sirtuin 1 superoxide dismutase TAR RNA-binding protein tert-butyl hydroperoxide exportin 5 zinc finger E-box-binding homeobox 1

Introduction Physiological reactive oxygen species (ROS) levels play an important role as second messengers within the intracellular signaling. ROS, which include superoxide anion, hydroxyl radicals, and hydrogen peroxide (H2O2), are generated as consequence of aerobic metabolism and are produced by several cellular sources. These include mitochondria, plasma membrane NADPH oxidase (NOX), and different

Aging https://doi.org/10.1016/B978-0-12-818698-5.00001-8

microRNA miRNAs are 21- to 23-nucleotide-long noncoding RNA molecules that modulate the stability and/or the

3

Copyright © 2020 Elsevier Inc. All rights reserved.

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1. Oxidative stress and miR-200c

translational efficiency of target messenger RNAs (mRNAs). miRNA genes can be expressed as polycistronic transcripts containing multiple miRNAs, as independent transcripts, or can be embedded in introns of protein coding mRNAs. Commonly miRNAs act as negative regulators of gene expression, although few opposite examples have been described.7 miRNA biogenesis (Fig. 1) starts with a primary transcript transcribed by RNA polymerase II, a generally thousands of nucleotides long mRNA termed the primiRNA; only few miRNAs are transcribed by RNA polymerase III. The pri-miRNA is a stem-loop structure containing the active miRNA. This hairpin undergoes nuclear cleavage by the ribonuclease III Drosha, complexed to the RNA-binding protein DGCR8/Pasha, to generate a 70–100 nucleotides pre-miRNA. Notably, most intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis; only a subset of intronic miRNAs, named mirtrons, enters in the

miRNA-processing pathway without a Drosha-mediated cleavage. Afterward the pre-miRNA is transported to the cytoplasm by the nuclear export factor exportin 5 (Xpo-5) and then processed by the ribonuclease III Dicer, complexed to TRBP (TAR RNA-binding protein), to form the mature 22-nt miRNA: miRNA* duplex. The mature single-stranded form is incorporated into the RNAinduced silencing complex (RISC), while the complementary strand miRNA* is typically degraded. Besides few exceptions, mammalian miRNAs base pair imperfectly with their mRNA targets and induce translational inhibition. Mature miRNAs loaded into the RISC mediate translational inhibition of target mRNAs through several different mechanisms. Moreover, RISC-miRNA complexes can shuttle mRNAs to specialized cytoplasmic compartments, the “P-bodies,” that are enriched in mRNA-catabolizing enzymes. Consequently, miRNAs also have an important effect on mRNA degradation. Since each miRNA can target

FIG. 1 miRNA biogenesis. RNA polymerase II transcribes miRNA genes to generate the primary transcripts (pri-miRNAs). The cleavage is mediated by the Drosha-DGCR8 complex, located in the nucleus. The
70-nucleotide-long product of the nuclear processing is a pre-miRNA, which shows a short stem plus
2-nucleotide 30 overhang. This structure is the signature motif, recognized by exportin 5 (Exp5), a nuclear export factor. Pre-miRNA constitutes a transport complex with Exp5 and its cofactor Ran. Ribonuclease III Dicer drives the second processing step (dicing) to produce miRNA duplexes in the cytoplasm. The duplex is then separated, and either of the strands is stably associated with RNA-induced silenced complex (RISC). The mature miRNA can inhibit the target genes by promoting translational repression and/or mRNA degradation.

1. Oxidative stress and aging

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miR-200c and oxidative stress

multiple transcripts and individual transcripts may be subject to multiple miRNA regulation, it may prove difficult to find a biological process or function that is not, at least in part, under the influence of miRNAs. The identification of miRNA targets is crucial to elucidate the role played by each miRNA in a biological function. The rules that guide miRNA/mRNA interactions are complex and still incompletely understood.8 The current paradigm states that a complete pairing between the 30 UTR region of the mRNA target and the “seed sequence” of the miRNA, a region centered on nucleotides 2–7, is required for miRNA-mRNA-mediated inhibition. Thus seed pairing is a necessary requirement for most target prediction algorithms. However, recent studies have demonstrated that also noncanonical miRNA binding can confer target regulation.9 Specifically, some mRNAs are targeted by miRNAs through recognition of 50 UTR or coding sequences. Moreover, “seedless” miRNA/mRNA interactions have been also demonstrated.10,11 The dysregulation of miRNAs has been linked to different conditions, including oxidative stress increase; in most cases the miRNA transcription is deregulated. In this respect, recent investigations demonstrated that epigenetic modifications such as histone acetylation or DNA methylation can play an important role.12

miR-200c and oxidative stress In a miRNA screening of ECs exposed to H2O2, the entire miR-200 family was induced by oxidative stress.13 The miR-200 family is composed of five members (miR200c, miR-141, miR-200a, miR-200b, and miR-429); in humans, miR-200c and miR-141 are clustered on chromosome 12, whereas miR-200a, miR-200b, and miR-429 are clustered on chromosome 1. They can also be TABLE 1

classified on the basis of the seed sequences: miR-200c, miR-200b, and miR-429 share the same seed sequence, whereas miR-200a and miR-141 show a different one. miR-200c is the most expressed family member and the most induced by oxidative stress in ECs; in fact, its expression increases nearly 30-fold upon 16 h treatment of H2O2, which is a very high upregulation for a microRNA; in fact, miRNAs are usually modulated around two- or threefolds.13 miR-141 also is highly induced, similarly to the cotranscribed miR-200c, and the other members are all induced albeit to a lower level (around two- to eightfold maximum). miR-200c upregulation upon H2O2 occurs also in murine myoblast, myotubes and in human fibroblast.13 Other interventions causing redox imbalance, such as the alkylating agent 1,3-bis(2 chloroethyl)1-nitrosourea (BCNU), a glutathione reductase inhibitor that blocks the conversion of oxidized to reduced glutathione, is also able to increase miR-200c expression. Moreover, BCNU-induced miR-200c increase is inhibited by the free-radical scavenger N-acetyl-L-cysteine (NAC), confirming the oxidative stress dependence of miR-200c upregulation.13 The upregulation of miR-200 family is also observed in different context and with different sources of oxidative stress summarized in Table 1; therefore it seems to be a conserved mechanism. miR-200c is upregulated by chronic oxidative stressinduced senescence in human fibroblasts and in human trabecular meshwork cells.14 miR-200c and miR-141 upregulation is observed in a cell model of oxidative stress by treatment of House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with different concentrations of tertbutyl hydroperoxide (t-BHP).15 miR-200 family induction following H2O2 exposure has been confirmed also in different cell lines, that is, mouse primary hippocampal neurons,16 human and mouse immortalized fibroblasts,

miR-200 family upregulation by different sources of ROS.

miRNA

ROS source

Tissue/organ

Source

Target

Functions

References

miR-200c, miR-141, miR-200a, miR-200b, miR-429

H2O2

Endothelium, myoblasts, fibroblasts

Human

ZEB1

Apoptosis/senescence

13

miR-200c

BCNU

Endothelium

Human

ZEB1

Apoptosis/senescence

13

miR-200c, miR-141, miR-200a, miR-200b, miR-429

H2O2

Colon carcinoma, melanoma cells, breast adenocarcinoma and ovarian

Human

p38

ROS accumulation; improved response to chemotherapy

17

miR-200c

H2O2

Primary hippocampal neurons

Mouse

Unknown

Unknown

16

miR-200c

chronic H2O2 treatment

Trabecular meshwork cells

Human

Unknown

Senescence

14

miR-200c, miR-141

t-BHP

Auditory cells

Mouse

Unknown

Unknown

15

1. Oxidative stress and aging

6

1. Oxidative stress and miR-200c

colon carcinoma, mammary gland epithelial cells and human cell lines, melanoma cells, kidney cells, breast adenocarcinoma, and ovarian adenocarcinoma.17 miR-141 and miR-200a, which display the same seed sequence, target p38α mitogen-activated protein kinase (MAPK). p38α is a signaling molecule that modulates cellular responses to stress18 and is involved in proliferation and survival control of many cell types.19 Indeed, p38α redox-sensing function is essential in the control of tumor development.20 Therefore enhanced expression of miR200 family miRNAs not only mimics p38α deficiency and increases tumor growth in mouse models but also improves the response to chemotherapeutic agents. In keeping the modulation of miR-200c and other miR-200 family expression has been exploited to sensitize different tumors to therapy.21–24

miR-200c induces apoptosis and senescence via ZEB1 inhibition The transcriptional factor zinc finger E-box-binding homeobox 1 (ZEB1) is a direct target of the entire miR-200 family and inhibits the transcription miR-200 family in a negative feedback loop.25 This loop has been investigated in the epithelial-mesenchymal transition (EMT) of many tumors, where miR-200 family expression levels decrease by determining the increase of their target

protein ZEB1, which is an inhibitor of the adhesion molecule E-cadherin. Therefore the decrease of miR-200 determines the lack of cell adhesion causing metastasis.25 The increase of miR-200c caused by oxidative stress in ECs downregulates ZEB1 that in nontumor cells elicits apoptosis and senescence, a state of permanent cell growth arrest in response to oxidative stress, recapitulating the oxidative stress-induced phenotype.13 Indeed the forced expression of ZEB1 in ECs overexpressing miR-200c partially rescued this phenotypes.13 The molecular mechanisms elicited by oxidative stress in ECs are depicted in Fig. 2. miR-200c upregulation by oxidative stress in ECs occurs at the transcriptional level, since H2O2 upregulates also the pre-miR-200c and this upregulation requires the tumor suppressors protein p53 and the retinoblastoma protein (pRb).13 Furthermore, ZEB1 transcription is under the control of pRb/E2F.26 In ECs, H2O2 induces a rapid pRb dephosphorylation via a serine-threonine phosphatase named PP2A27,28; this allows pRb binding and inhibition of the E2F transcriptional activity factor on ZEB1 gene.26 Therefore, upon oxidative stress, the upregulation of miR-200c inhibits ZEB1 protein translation and induces ZEB1 mRNA degradation; in addition, ZEB1 mRNA decreases because of a pRb/E2F-dependent mechanism. ZEB1 demise induces a further upregulation of miR-200c, since it is a transcriptional inhibitor of miR200c,25 reinforcing the upregulation of miR-200c.13

FIG. 2 miR-200c upregulation in ECs occurs via p53- and pRb-dependent mechanisms. H2O2 induces a rapid dephosphorylation of pRb via a serine-threonine phosphatase (PP2A)-dependent mechanism, repressing transcription factor E2F activity. ZEB1 is under pRb/E2F control. Therefore H2O2 dephosphorylating pRb causes ZEB1 mRNA and protein level downregulation. ZEB1 is a transcriptional inhibitor of miR-200c; consequently, its decrease provokes miR-200c upregulation. miR-200c transcriptional upregulation is also induced by p53, which is induced by oxidative stress. p53 in turn induces p21, which sustains pRb dephosphorylation, further decreasing ZEB1. ZEB1 downregulation induces p21 transcription, being ZEB1 a transcriptional inhibitor of p21. Finally, ZEB1 demise provokes growth arrest, senescence, and apoptosis.

1. Oxidative stress and aging

miR-200c and oxidative stress

7

Finally, oxidative stress induces p53 activity that is known to induce the cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 (p21) transcription that reinforce pRb dephosphorylation.29 Additionally, p53 induces apoptosis and miR-200c transcription, enhancing miR-200c increase. Finally, ZEB1 is a transcriptional inhibitor of p2130; therefore its downregulation further increases p21. This molecular circuitry induces growth arrest, apoptosis, and senescence, which are all the effects induced by oxidative stress (Fig. 2).

miR-200c oxidative stress and endothelial dysfunction A fundamental molecular circuit for vascular homeostasis is the one existing among sirtuin 1 (SIRT1), endothelial nitric oxide synthase (eNOS), and forkhead box O1 (FOXO1) proteins. These proteins play a key role in endothelial function, since they are involved in oxidative stress resistance (Fig. 3). SIRT1 plays an important role in cell senescence, having an antiinflammatory and antioxidant role.31 Indeed, it is known that ROS and aging cause a reduction in the expression of SIRT1, causing senescence and endothelial dysfunction, and the activation of SIRT1 improves the response to oxidative stress.32 Furthermore, SIRT1 promotes mitochondrial biogenesis and NO production released by eNOS.31,32 In turn, NO increases the stability of mRNA and SIRT1 protein, confirming the existence of a positive circuit between eNOS activity and SIRT1 expression levels.32 In the molecular circuit existing between eNOS and SIRT1, the transcription of FOXO1 plays a very important role. In fact, FOXO1 is a direct target of SIRT1 deacetylation; deacetylation of FOXO1 induces its transcriptional activity on the promoter of SIRT1 and of CAT and manganese superoxide dismutase (MnSOD) antioxidant enzymes.32 The correct functioning of the SIRT1/eNOS/FOXO1 molecular circuit plays a fundamental role in endothelial survival and in vasodilation (Fig. 3). An important player in oxidative stress response and modulation is the p66 isoform of ShcA protein (p66Shc), a key regulator of mitochondrial ROS production by a variety of different stimuli.33 Oxidative stress induces the phosphorylation of p66Shc protein in serine 36 residue (Fig. 3), which causes an increase in ROS in three cellular districts: - in the nucleus, where it inhibits FOXO134; - in the mitochondria, where it binds to cytochrome c, acting as an oxidoreductase enzyme that generates ROS35; - in the plasma membrane, where it activates NADPH oxidase.36

FIG. 3 miR-200c disrupts the regulatory loop existing among eNOS/ SIRT1/FOXO1. miR-200c inhibits situin1 (SIRT1)/endothelial nitric oxide synthase (eNOS)/forkhead box O1 (FOXO1) regulatory loop, by targeting directly all of them. This loop controls different cell functions. Specifically, miR-200c increases ROS production by two mechanisms: (i) it decreases ROS scavengers by targeting peroxiredoxin 2 (PRDX2) and forkhead box O1 (FOXO1), a transcription factor required for catalase (CAT) and manganese superoxide dismutase (MnSOD) expression; (ii) it sustains ROS production via p66Shc phosphorylation in serine 36. miR-200c decreases NO production by targeting eNOS. miR-200c induces senescence targeting SIRT1 deacetylase that causes the following: (i) eNOS acetylation increase further reducing the bioavailability of NO that is a molecule that stabilizes SIRT1 mRNA and protein, thus provoking a further SIRT1 decrease, and (ii) acetylation of FOXO1 increase causing FOXO1 transcriptional activity inhibition. In addition, miR-200c targets FOXO1 both directly and indirectly via a p66Shc phosphorylation-dependent mechanism that inhibits FOXO1 transcriptional activity, decreasing CAT and MnSOD expression, further sustaining ROS production. In conclusion, ROS-induced miR200c disrupts SIRT1/FOXO1/eNOS loop promoting cell senescence and activating a complex molecular pathway that sustains oxidative stress and miR-200c upregulation.

miR-200c has been shown to target directly SIRT1, eNOS, and FOXO137; ROS-upregulated miR-200c decreases NO and increases the acetylation of SIRT1 protein targets, such as FOXO1 and p53. p53 acetylation increases its transcriptional activity on miR-200c and also its ability to induce senescence and apoptosis. FOXO1 acetylation inhibits its transcriptional activity, decreasing the expression of antioxidant enzymes such as CAT and MnSOD, therefore increasing ROS production. Consequently, miR-200c increases ROS and induces the phosphorylation of p66Shc protein in Ser-36, which in turn inhibits the transcriptional activity of FOXO1, reinforcing this molecular circuit (Fig. 3).

1. Oxidative stress and aging

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1. Oxidative stress and miR-200c

Moreover, miR-200c has been demonstrated to target directly also peroxiredoxin 2,38 a selective scavenger for H2O2,39 further inducing oxidative stress. Aging is a process of functional deterioration of an organism that can occur at different levels (cellular, tissue, and organelle), bringing life to end. One of the most relevant players in the aging process and agerelated disorders is cellular senescence. In humans and in many animal models, an age-dependent increase in oxidative stress has been demonstrated, showing that increased ROS can be both a consequence and a cause of aging. In keeping, the in vitro results obtained in ECs are also recapitulated in different in vivo oxidative stress models, namely, in human skin fibroblasts from elderly donors, femoral arteries of old mice, and in a mouse model of ischemia of the hind limbs.37 In all cases, miR-200c was higher than the control, and its targets, that is, SIRT1, eNOS, and FOXO1, were decreased. In the mouse model of hind limb ischemia, treatment with anti-miR-200c restored the expression of these proteins and limb perfusion.37 Aging increases miR-200c expression also in other tissues different from the aforementioned described and all summarized in Table 2. These include skeletal muscle from rhesus monkeys40 and human liver.41 TABLE 2 miR-200c upregulation in aged tissues. Tissue/organ

Source

References

Skeletal muscle

Monkey

40

Liver

Human

41

Skin fibroblast

Human

37

Femoral artery

Mouse

37

Applications to other diseases or conditions miR-200c is increased in different pathological conditions associated to an increase of oxidative stress (summarized in Table 3) such as acute hind limb ischemia in skeletal muscle,13,37 ischemia and ischemia/reperfusion in the brain,42 in different tissues in diabetes,43–47 high glucose-induced cardiac hypertrophy,48 nonalcoholic steatohepatitis,49 and Duchenne muscle dystrophy.50 Moreover, miR-200c and the entire miRNA family are deregulated in many different types of cancers, since they are deeply involved in the EMT of tumor cells.51 In many of the previously described disease conditions, the inhibition of miR-200c was able to recover most of thedeleterious effects caused by miR-200c increase.37,48–50 miR-200c expression is also increased in cardiotoxicity induced by anthracyclines, such as doxorubicin (DOX) treatment in mice and in human cardiac mesenchymal progenitor cells.52 Furthermore, circulating miR-200c levels are increased in children with familial hypercholesterolemia53 and in adult patients with carotid artery plaques,54 both conditions associated with enhanced ROS. Moreover, miR-200c increases also in atherosclerotic carotid plaques in human patients, and it is more enhanced in unstable than stable plaques.54 In conclusion, miR-200c increases in an age-dependent manner and in different pathological conditions that display an increase of ROS. miR-200c upregulation causes a decrease of antioxidants, an increase of ROS, and a decrease of NO, all features associated with aging. Therefore the comprehension of miR-200c roles and targets may support the analysis of new therapeutic strategies to delay and treat age-related modifications and aging signs.

TABLE 3 miR-200c modulation in diseases. Disease

Regulation

Tissue/organ

Source

References

Cancer

Down/up

Different tissues/organs

Human

51

Ischemia

Up

Skeletal muscles

Mouse

13,37

Ischemia/reperfusion

Up

Brain

Mouse

42

Cardiac hypertrophy

Up

Cardiomyocytes

Rat

48

Nonalcoholic steatohepatitis

Up

Liver

Human

49

Duchenne muscle dystrophy

Up

Different muscles, myoblasts

Mouse, Human

50

Familial hypercholesterolemia

Up

Plasma

Human

53

Atherosclerosis

Up

Carotid arteries, plasma

Human

54

Diabetes

Up

Heart, VSMC, pancreatic beta cells, renal arteries

Mouse/human

43–47

1. Oxidative stress and aging

9

References

Summary points • ROS play a causative pathological role in different cellular processes implicated in growth arrest, senescence, and cell death. The cells have developed a series of enzymatic and nonenzymatic antioxidant defenses to maintain the intracellular redox state homeostasis. miRNAs are short noncoding RNAs that modulate the stability and/or the translational efficiency of target messenger. Each miRNA can regulate multiple mRNAs, and each mRNA can be regulated by many miRNAs. miRNAs can act as key regulators in cell proliferation and cell death, early development, apoptosis, metabolism, and cell differentiation. Dysregulation of miRNAs has been associated with different diseases. • miR-200 family is induced by different sources of oxidative stress and in many different nontumor and tumor cells. miR-200 family is composed of five members: miR200a, miR-200b, miR-200c, miR-141, and miR-429. miR-200c is the most upregulated family member in ECs upon H2O2 treatment. miR-200c upregulation by H2O2 in ECs is transcriptional and involves p53 and pRb. ZEB1 is a direct target of the entire miR-200 family and inhibits the transcription of miR-200 family in a negative feedback loop. ZEB1 plays important functions in the tumor progression process and in metastatic diffusion inhibiting E-cadherin. ZEB1 demise induced by oxidative stress in ECs is caused by miR-200c- and pRb/E2F-dependent mechanisms. ZEB1 downregulation in nontumor cells is responsible for cell growth arrest, apoptosis, and senescence induction. • The loop existing among SIRT1, eNOS, and FOXO1 is a molecular regulatory circuit involved in the maintenance of vascular homeostasis. These proteins are involved in resistance to oxidative stress and play fundamental roles in EC survival and vasodilation. miR-200c destroys the loop existing between SIRT1, eNOS, and FOXO1 targeting all of them. miR-200c disrupting this loop increases ROS and decreases NO, causing endothelial dysfunction. Moreover, miR-200c enhances oxidative stress inducing p66Shc protein phosphorylation in Ser36 that sustains ROS increase. miR-200c is upregulated in different pathophysiological conditions associated with an increase of ROS including, aging, diabetes, nonalcoholic steatohepatitis, diabetic cardiac

hypertrophy, hind limb ischemia, brain ischemia reperfusion, Duchenne muscle dystrophy, familial hypercholesterolemia, and atherosclerosis.

References 1. Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol 2003;15:247–54. 2. Fridovich I. The biology of oxygen radicals. Science 1978;201:875–80. 3. Yang YM, Huang A, Kaley G, Sun D. eNOS uncoupling and endothelial dysfunction in aged vessels. Am J Physiol Hear Circ Physiol 2009;297:H1829–36. 4. Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest 2003;112:725–35. 5. Pani G, Koch OR, Galeotti T. The p53-p66shc-manganese superoxide dismutase (MnSOD) network: a mitochondrial intrigue to generate reactive oxygen species. Int J Biochem Cell Biol 2009;41:1002–5. 6. Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP, Lanfrancone L, Pelicci PG. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 1999;402:309–13. 7. Vasudevan S. Posttranscriptional upregulation by microRNAs. Wiley Interdiscip Rev RNA 2012;3:311–30. 8. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215–33. 9. Elefant N, Altuvia Y, Margalit H. A wide repertoire of miRNA binding sites: prediction and functional implications. Bioinformatics 2011;27:3093–101. 10. Fasanaro P, Romani S, Voellenkle C, Maimone B, Capogrossi MC, Martelli F. ROD1 is a seedless target gene of hypoxia-induced miR-210. PLoS One 2012;7:e44651. 11. Lal A, Navarro F, Maher CA, et al. miR-24 inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to “seedless” 3’UTR microRNA recognition elements. Mol Cell 2009;35:610–25. 12. Wang Z, Yao H, Lin S, Zhu X, Shen Z, Lu G, Poon WS, Xie D, Lin MC, Kung H. Transcriptional and epigenetic regulation of human microRNAs. Cancer Lett 2013;331:1–10. 13. Magenta A, Cencioni C, Fasanaro P, Zaccagnini G, Greco S, SarraFerraris G, Antonini A, Martelli F, Capogrossi MC. miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition. Cell Death Differ 2011;18:1628–39. 14. Li G, Luna C, Qiu J, Epstein DL, Gonzalez P. Alterations in microRNA expression in stress-induced cellular senescence. Mech Ageing Dev 2009;130:731–41. 15. Wang Z, Liu Y, Han N, Chen X, Yu W, Zhang W, Zou F. Profiles of oxidative stress-related microRNA and mRNA expression in auditory cells. Brain Res 2010;1346:14–25. 16. Xu S, Zhang R, Niu J, Cui D, Xie B, Zhang B, Lu K, Yu W, Wang X, Zhang Q. Oxidative stress mediated-alterations of the microRNA expression profile in mouse hippocampal neurons. Int J Mol Sci 2012;13:16945–60. 17. Mateescu B, Batista L, Cardon M, et al. miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response. Nat Med 2011;17:1627–35. 18. Dolado I, Swat A, Ajenjo N, De Vita G, Cuadrado A, Nebreda AR. p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis. Cancer Cell 2007;11:191–205. 19. Hui L, Bakiri L, Mairhorfer A, Schweifer N, Haslinger C, Kenner L, Komnenovic V, Scheuch H, Beug H, Wagner EF. p38alpha suppresses normal and cancer cell proliferation by antagonizing the JNK-c-Jun pathway. Nat Genet 2007;39:741–9.

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20. Kennedy NJ, Cellurale C, Davis RJ. A radical role for p38 MAPK in tumor initiation. Cancer Cell 2007;11:101–3. 21. Kopp F, Oak PS, Wagner E, Roidl A. miR-200c sensitizes breast cancer cells to doxorubicin treatment by decreasing TrkB and Bmi1 expression. PLoS One 2012;7:e50469. 22. Liu Y, Zhu S-T, Wang X, Deng J, Li W-H, Zhang P, Liu B-S. MiR-200c regulates tumor growth and chemosensitivity to cisplatin in osteosarcoma by targeting AKT2. Sci Rep 2017;7:13598. 23. Cortez MA, Valdecanas D, Zhang X, et al. Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer. Mol Ther 2014;22:1494–503. 24. Cittelly DM, Dimitrova I, Howe EN, et al. Restoration of miR-200c to ovarian cancer reduces tumor burden and increases sensitivity to paclitaxel. Mol Cancer Ther 2012;11:2556–65. 25. Brabletz S, Brabletz T. The ZEB/miR-200 feedback loop—a motor of cellular plasticity in development and cancer? EMBO Rep 2010;11:670–7. 26. Liu Y, Costantino ME, Montoya-Durango D, Higashi Y, Darling DS, Dean DC. The zinc finger transcription factor ZFHX1A is linked to cell proliferation by Rb-E2F1. Biochem J 2007;408:79–85. 27. Cicchillitti L, Fasanaro P, Biglioli P, Capogrossi MC, Martelli F. Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb, p107, and p130. J Biol Chem 2003;278:19509–17. 28. Magenta A, Fasanaro P, Romani S, Di Stefano V, Capogrossi MC, Martelli F. Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation. Mol Cell Biol 2008; 28:873–82. 29. Liu D, Xu Y. p53, oxidative stress, and aging. Antioxid Redox Signal 2011;15:1669–78. 30. Liu Y, El-Naggar S, Darling DS, Higashi Y, Dean DC. Zeb1 links epithelial-mesenchymal transition and cellular senescence. Development 2008;135:579–88. 31. Chen Z, Shentu T-P, Wen L, Johnson DA, Shyy JY-J. Regulation of SIRT1 by oxidative stress-responsive miRNAs and a systematic approach to identify its role in the endothelium. Antioxid Redox Signal 2013;19:1522–38. 32. Potente M, Dimmeler S. Emerging roles of SIRT1 in vascular endothelial homeostasis. Cell Cycle 2008;7:2117–22. 33. Giorgio M, Trinei M, Migliaccio E, Pelicci PG. Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals? Nat Rev Mol Cell Biol 2007;8:722–8. 34. Nemoto S, Finkel T. Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway. Science 2002;295:2450–2. 35. Giorgio M, Migliaccio E, Orsini F, et al. Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell 2005;122:221–33. 36. Khanday FA, Yamamori T, Mattagajasingh I, et al. Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress. Mol Biol Cell 2006;17:122–9. 37. Carlomosti F, D’Agostino M, Beji S, et al. Oxidative stress-induced miR-200c disrupts the regulatory loop among SIRT1, FOXO1 and eNOS. Antioxid Redox Signal 2016;27(6):328–44. https://doi.org/ 10.1089/ars.2016.6643. 38. Shi L, Zhang S, Wu H, Zhang L, Dai X, Hu J, Xue J, Liu T, Liang Y, Wu G. MiR-200c increases the radiosensitivity of non-small-cell lung cancer cell line A549 by targeting VEGF-VEGFR2 pathway. PLoS One 2013;8:e78344.

39. Peskin AV, Low FM, Paton LN, Maghzal GJ, Hampton MB, Winterbourn CC. The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents. J Biol Chem 2007;282:11885–92. 40. Mercken EM, Majounie E, Ding J, et al. Age-associated miRNA alterations in skeletal muscle from rhesus monkeys reversed by caloric restriction. Aging (Albany NY) 2013;5:692–703. 41. Capri M, Olivieri F, Lanzarini C, et al. Identification of miR-31-5p, miR-141-3p, miR-200c-3p, and GLT1 as human liver aging markers sensitive to donor–recipient age-mismatch in transplants. Aging Cell 2017;16(2):262–72. https://doi.org/10.1111/acel.12549. 42. Lee S-T, Chu K, Jung K-H, et al. MicroRNAs induced during ischemic preconditioning. Stroke 2010;41:1646–51. 43. Belgardt B-F, Ahmed K, Spranger M, et al. The microRNA-200 family regulates pancreatic beta cell survival in type 2 diabetes. Nat Med 2015;21:619–27. 44. Klein D, Misawa R, Bravo-Egana V, et al. MicroRNA expression in alpha and beta cells of human pancreatic islets. PLoS One 2013;8: e55064. 45. Baseler WA, Thapa D, Jagannathan R, Dabkowski ER, Croston TL, Hollander JM. miR-141 as a regulator of the mitochondrial phosphate carrier (Slc25a3) in the type 1 diabetic heart. Am J Physiol Cell Physiol 2012;303:C1244–51. 46. Reddy MA, Jin W, Villeneuve L, Wang M, Lanting L, Todorov I, Kato M, Natarajan R. Pro-inflammatory role of microrna-200 in vascular smooth muscle cells from diabetic mice. Arter Thromb Vasc Biol 2012;32:721–9. 47. Zhang H, Liu J, Qu D, et al. Inhibition of miR-200c restores endothelial function in diabetic mice through suppression of COX-2. Diabetes 2016;65(5):1196–207. https://doi.org/10.2337/db15-1067. 48. Singh GB, Raut SK, Khanna S, Kumar A, Sharma S, Prasad R, Khullar M. MicroRNA-200c modulates DUSP-1 expression in diabetes-induced cardiac hypertrophy. Mol Cell Biochem 2017;424:1–11. 49. Tran M, Lee S-M, Shin D-J, Wang L. Loss of miR-141/200c ameliorates hepatic steatosis and inflammation by reprogramming multiple signaling pathways in NASH. JCI Insight 2017;2(21). https://doi.org/10.1172/jci.insight.96094. 50. D’Agostino M, Torcinaro A, Madaro L, et al. Role of miR-200c in myogenic differentiation impairment via p66Shc: implication in skeletal muscle regeneration of dystrophic mdx mice. Oxid Med Cell Longev 2018;2018:4814696. 51. Park S-M, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 2008;22:894–907. 52. Beji S, Milano G, Scopece A, et al. Doxorubicin upregulates CXCR4 via miR-200c/ZEB1-dependent mechanism in human cardiac mesenchymal progenitor cells. Cell Death Dis 2017;8(8):e3020. https://doi.org/10.1038/cddis.2017.409. 53. D’Agostino M, Martino F, Sileno S, et al. Circulating miR-200c is up-regulated in paediatric patients with familial hypercholesterolaemia and correlates with miR-33a/b levels: implication of a ZEB1-dependent mechanism. Clin Sci (Lond) 2017;131:2397–408. 54. Magenta A, Sileno S, D’Agostino M, Persiani F, Beji S, Paolini A, Camilli D, Platone A, Capogrossi MC, Furgiuele S. Atherosclerotic plaque instability in carotid arteries: miR-200c as a promising biomarker. Clin Sci (Lond) 2018;132:2423–36.

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C H A P T E R

2 Caloric restriction, reactive oxygen species, and longevity Miyuki Kobara, Hiroe Toba,Tetsuo Nakata Department of Clinical Pharmacology, Division of Pathological Science, Kyoto Pharmaceutical University, Kyoto, Japan

Adequate CR extends healthy life span, whereas excessive CR with malnutrition is harmful. Therefore, CR to an appropriate extent and for a sufficient duration to maximize healthy life span is important. Treatment costs for age-related diseases are now markedly increasing in the aging society, and thus the cost-effective prevention of age-related diseases is desired. CR has favorable cost benefits. In this chapter, we summarize the effects of CR in a number of organisms and describe the main proposed signaling pathways for the beneficial effects of CR, with a focus on oxidative stress.

List of abbreviations AMPK CR FoxO GPx hsCRP IGF-1 IL-1β NFkβ PGC1-α ROS SIRT1 SOD TNF-α TOR

adenosine 30 ,50 -monophosphate-dependent protein kinase caloric restriction forkhead transcription factor O glutathione peroxidase high-sensitivity C-reactive protein insulin growth factor 1 interleukin-1β nuclear factor kappa β peroxisome proliferator-activated receptor-γ coactivator 1-α reactive oxygen species silent information regulator 1 superoxide dismutase tumor necrosis factor α target of rapamycin

CR in animals

Introduction

CR was initially reported by McCay in 1935, who showed that a reduced food intake extended the life span of rats.6 Subsequent studies in this field confirmed favorable life span elongation by CR in many organisms from worms to nonhuman primates.2 CR also retarded agerelated cancers, cardiovascular diseases, stroke, diabetes, dyslipidemia, and neural degenerative diseases in these short-lived organisms.7 These age-related diseases are major causes of mortality, and neural disorders decrease the quality of life and shorten the health span of the elderly. As a bridge from these short-lived animals to humans, prospective examinations on the effects of CR in rhesus monkeys were initiated by two independent associations in the United States from the late 1980s: the Wisconsin National Primate Research Center based at the University of Wisconsin-Madison (WNPRC) study and the National Institute on Aging (NIA) study. In the WNPRC study, 30% CR in adult rhesus monkeys significantly delayed age-associated pathological conditions and improved all-cause mortality.8 On the other hand, in the NIA study,

Aging is inevitable and progressive phenomena in all living organisms. The accumulation of damage leads to functional decline and structural deterioration in organs in association with increases in susceptibility to diseases, ultimately resulting in death as the final stage. Agerelated diseases, such as cancer, cardiovascular diseases, stroke, and neurally degenerative diseases, are now the leading causes of mortality worldwide.1 In antiaging research conducted to date, numerous interventions to retard the aging process have been examined. Among antiaging interventions, caloric restriction (CR), a reduction in caloric intake without malnutrition, is a powerful nongenetic intervention that prolongs maximum and mean life spans and suppresses age-related diseases.2–4 These favorable phenomena are conserved in many species from yeast to mammals.2–5 Therefore, elucidation of the mechanism responsible for the beneficial effects of CR may reveal targets for age-related disease retardation and healthy life span elongation.

Aging https://doi.org/10.1016/B978-0-12-818698-5.00002-X

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Copyright © 2020 Elsevier Inc. All rights reserved.

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2. Caloric restriction and longevity

all-cause mortality did not improve in CR monkeys even though similar CR was conducted; however, measurements on the metabolic health index and overall function showed that old-onset CR exerted beneficial effects.9 Although the impact of CR on mortality differs, comparisons of important evidence from these long-term studies demonstrated that CR resulted in a favorable lipid profile and reduced susceptibility to cancer, type 2 diabetes, and cardiovascular diseases.10

Mechanisms responsible for beneficial effects of CR in animals Oxidative stress and inflammation In the aging process, tissue and cellular damage induced by oxidative stress and inflammation accumulate.11 Oxidative stress also enhances the progression of age-related diseases, including cancer, neurodegeneration, cardiovascular diseases, and diabetes.12 CR studies using animal models and humans demonstrated that CR reduced the production of ROS, resulting in decreases in lipid peroxidation, protein carbonylation, and DNA/ RNA oxidation, and also increased the antioxidant capacity, including glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase.7,12–16 CR was shown to suppress the age-related and oxidative stress-related diseases of cancer, neurodegeneration, and cardiovascular diseases.12 It also inhibited inflammation and reduced the levels of inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β).2 Among several sources of ROS production in the aging process, damaged mitochondria are important.17 Mitochondrial ROS were shown to induce mitochondrial DNA mutations, leading to an impaired mitochondrial respiratory chain, while a deficiency in mtDNA polymerase promoted premature aging in mammals.18 Previous studies found that CR attenuated mitochondrial ROS production from the respiratory chain and preserved its function.7,19,20 Therefore, life span elongation by CR may be due to, at least in part, antioxidant activities and the preservation of mitochondrial function.19 However, the importance of antioxidants for CR-induced life span elongation remains controversial. One of the reasons for this is the absence of an effect of the deletion of NF-E2-related factor 2, a well-known transcription factor of antioxidant enzymes, on CR-induced life span elongation.21 Moreover, Wang et al. reported that CR-induced mild oxidative stress by mitochondria plays positive and integrative roles as adaptive phenomena, which are beneficial rather than detrimental in the aging process.17 Previous studies reported that CR-induced ROS also slowed aging

and prevented the progression of cancer.22 Therefore, the roles of ROS in the effects of CR may be beneficial and detrimental depending on their source and volumes.

Insulin/IGF-1 pathway During nutrient restriction, growth signaling by the insulin/IGF-1 pathway is suppressed as an adaptation,2 and animal models of the genetic suppression of the insulin/IGF-1 pathway showed an extended life span.2,23 The CR-induced suppression of the insulin/IGF-1 signaling activates the forkhead family of transcription factors (FoxO) through phosphatidylinositol-3-kinase (PI3K) and Akt inhibition.24,25 Since FoxO3 is a transcription factor of several antioxidant genes,26 this signaling pathway contributes to the CR-induced inhibition of oxidative stress. However, the contribution of this signaling pathway to CR-induced life span elongation has not been fully clarified, because CR could elongate life span in genetic knockout animals of IGF-1 signaling.2 Therefore, the insulin/ IGF-1 signaling pathway is enhanced during CR and contributes to its beneficial effects; however, the dependency of CR-induced life span elongation on this signaling pathway is limited.

The TOR pathway and autophagy The TOR signaling pathway is another nutrient sensor that is suppressed by CR.27 As another candidate pathway for CR-induced longevity, the suppression of TOR signaling has been confirmed, and its deletion has been shown to extend the life span of many organisms.2,27 TOR suppression under nutrient starvation conditions enhanced autophagy, which removes damaged mitochondria, a major source of ROS production, and preserves their function.28,29

The AMPK pathway AMPK is an energy sensor of nutritional starvation. CR activates AMPK in association with declines in the AMP/ATP ratio in many organisms, and the deletion of AMPK blunts CR-induced life span elongation.30 AMPK regulates many intracellular signaling pathways, including the activation of FoxO,30 SIRT1, peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α), and the suppression of TOR.4 These factors collaborate with the suppression of oxidative stress. Another pathway for AMPK-mediated ROS reductions is the preservation of NADPH levels through the suppression of acetyl CoA carboxylases.31

1. Oxidative stress and aging

13

CR in humans

Sirtuins Sirtuins are a family of NAD+-dependent protein deacetylase that regulate the activities of many enzymes by deacetylation. Under nutrient restriction states, sirtuins are activated as nutrient sensors in association with increments in the NAD+/NADH ratio and mediate CR-induced life and health span elongation.32,33 In a mouse model the attenuation of aging-related hearing loss by CR was blunted in SIRT3 deletion mice.34 In mammals, CR has also been shown to activate sirtuins, particularly SIRT1, in many organs, leading to an enhanced antioxidant capacity by FoxO3.26,33 In addition, CR activated mitochondrial SIRT3, which preserved the activity of Mn-SOD35 and enhanced the mitochondrial glutathione antioxidant defense system, leading to mitochondrial protection.36

Mitochondrial maintenance In the aging process the abundance of mitochondrial DNA and its functions were found to be impaired in human skeletal muscle.37 Mitochondria are central to energy metabolism in cells and nutrient restriction induces metabolic reprogramming in mitochondrial energy metabolism. The AMPK/PGC1-α pathway and mitochondrial sirtuin SIRT 3 play critical roles in maintaining mitochondrial function, and both signaling pathways were shown to be activated by CR.30,33 The genetic deletion of SIRT3 abolished protection of mitochondria by CR.4 Changes in membrane lipid compositions and peroxidation, which are involved in the aging process,38 were recently shown to regulate mitochondrial function and affect CR-induced longevity.39 Collectively,

these finding show that mitochondrial maintenance and biogenesis play important roles in the beneficial effects of CR. In summary, multiple signal transduction pathways are activated in nutritional restriction, and cross talk among each signaling pathway influences the beneficial effects of CR. Since the majority of these signaling pathways reduce oxidative stress, the suppression of ROS production and protection of mitochondria by CR have critical roles in life span elongation and the retardation of age-related diseases. On the other hand a small amount of ROS functions as a trigger for mitochondrial maintenance and has beneficial roles (Fig. 1).

CR in humans The most important question in this research field is whether the beneficial effects of CR, such as life and health span elongation, are conserved and if CR is feasible and safe in humans. However, the human life span is long, and the strict restriction of nutrient intake for a long period is very difficult. Therefore direct evidence for the beneficial effects of CR on life span in humans is difficult to obtain. Therefore the majority of studies being conducted on humans in the CR research field involve the effects of short-term CR on candidates, which are assumed to achieve life span elongation in animal models. The first human study on CR was performed by Strom et al. in 1951.40 In this study, Scandinavians were placed on a semistarvation diet in the 1940s as a consequence of World War II, and this was associated with a reduction in the prevalence of heart diseases.40 Several clinical trials

CR Trigger

fasting

glucose

Insulin/ IGF-1 signaling Effector, Signaling pathway

AMP/ATP

S6K Protein synthesis

Sirtuins SIRT1 SIRT3

AMPK

TOR

Akt

NAD+/NADH

autophagy

PCG1a

ACC Remove damaged organelle

Mn-SOD

Mitochondrial Mitochondrial maintenance biogenesis

FoxO NADH

Oxdative stress

1. Oxidative stress and aging

FIG. 1 Relationships between signal transduction in CR and oxidative stress. A number of nutrient sensors are modified in CR and contribute to CR-induced life span elongation and the retardation of age-related disorders in organisms.

14

2. Caloric restriction and longevity

were subsequently performed. The main randomized clinical trials on human CR were conducted in the United States in the early 21st century. Three independent pilot studies (comprehensive assessment of the long-term effects of reducing intake of energy [CALERIE]-1) were performed at Tufts University, Washington University, and the Pennington Biomedical Research Center, using different protocols. To confirm the long-term feasibility and effectiveness of CR, a multicenter single protocol study was also conducted (CALERIE-2) (Table 1).

TABLE 1

Summary of the design and main findings from CALERIE-1 and -2 studies, major human clinical studies of CR. CALERIE-1

CALERIE-2

Tafts

Washington

PBRC

Duration

1Y

1Y

6M

2Y

Age

25–42

50–60

25–50(F45)

21–50 (F47)

BMI

25–29.9

23.5–29.9

25–29.9

22–28

CR

30% CR + HG

20% CR

25% CR

30% CR + LG

20% Ex

12.5% CR + 12.5% Ex

10% CR + HG

WD

LCD

#!"

10% CR + LG

Control

Control

BW

#

#

#

#

Fat mass

#

#

#

#

!

!

#

BP Lipid profile

TC#

TC#

LDL#

LDL#

HDL "

HDL!

Insulin resistance Oxidative stress

TC#

HOMA-IR #

HOMA-IR# GPx "

DNA damage#

Protein carbonyl# SOD! Catalase!

Protein carbonyl!

F2isoprostane #

Data from Das SK, Balasubramanian P, Weerasekara YK. Nutrition modulation of human aging: the calorie restriction paradigm. Mol Cell Endocrinol 2017;455:148–57. Il’yasova D, Fontana L, Bhapkar M, Pieper CF, Spasojevic I, Redman LM, Das SK, Huffman KM, Kraus WE, CALERIE Study Investigators. Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2-isoprostanes: the CALERIE 2 randomized clinical trial. Aging Cell 2018;17:e12719, with permission from Publishers.

CALERIE-1 study In the CALERIE-1 study conducted at Tufts University, 30% CR in obese individuals for 1 year significantly reduced body weight, improved glucose-insulin dynamics, increased the plasma GPx capacity, an antioxidant, and decreased protein carbonyl levels, an indicator of oxidative stress.41 On the other hand, SOD and catalase levels were not affected by this CR.41 However, 30% CR was not feasible for more than 1 year. In the CALERIE-1 study conducted at Washington State University, nonobese individuals were included and separated into two groups: 20% dietary CR (CR group) or 20% increased energy expenditure by exercise (EX group) for 1 year. Energy reductions were similar between the CR and EX groups. Insulin sensitivity, blood pressure, and lipid profiles showed similar improvements in the EX and CR groups,42,43 and DNA and RNA oxidation in white blood cells was reduced.44 In the CALERIE-1 study conducted at the Pennington Biomedical Research Center (PBRC), overweight individuals were enrolled. Participants in the CR alone group (25% reduction in nutritional caloric intake alone) and CR + EX group (mild nutritional caloric reduction [12.5%] with 12.5% increase in energy expenditure by exercise) showed a lower core body temperature, a previously reported index of longevity, reduced body weight, and a favorable lipid profile and insulin sensitivity.5 Furthermore, the expression of mitochondrial functionpreserving genes, such as PGC1-α, TFAM, and SIRT1, and the mitochondrial DNA content in skeletal muscle were preserved in both CR groups.45

CALERIE-2 study After these three pilot studies, the CALERIE-2 trial was performed for 2 years using a single protocol at three sites. Nonobese participants were divided into a 25% CR group and ad libitum caloric intake control group. In the CALERIE-2 study, although planned CR was 25%, the real average restriction level was 11.7  0.7%, which was feasible and sustainable.5,46 CR significantly reduced body weight and mean blood pressure, in association with a favorable lipid profile and insulin sensitivity, indicating the attenuation of cardiovascular risks.46,47 On the other hand a reduced core body temperature was not observed in the CR group.47 Serum TNF-α and high-sensitivity C-reactive protein (hsCRP); indexes of inflammation; and urine F2-isoprostanes, a marker of oxidative stress, were significantly lower in the CR group.5,14,46 On the other hand, in muscle biopsy samples, mitochondrial biogenesis and oxidative stress markers were unaffected.48

1. Oxidative stress and aging

Trials on other modified CR and other modules mimicking CR-induced beneficial signaling

ROS CVD Cancer DM Bone fracture

15

FIG. 2 Proposed scheme for CR and alternative CR methods for longevity, age-related diseases, and ROS. ROS are gradually increased with aging in association with age-related diseases, such as cardiovascular diseases, cancer, diabetes mellitus, and bone fracture. CR and alternative CR attenuate these age-related disorders and result in life and health span elongation.

Nutrient component restriction

CR

ROS CVD Cancer DM Bone fracture

Recent CR studies in humans After these long clinical trials, the effects of short-term CR for 4 weeks on oxidative stress in obese individuals were examined. Four-week CR reduced the systemic oxidative stress markers of protein carbonyl and F2-isoprostane levels, and activated antioxidant enzymes, including SOD and GPx.49,50 Furthermore, in recent studies by LopezDomenech et al.,15,16 obese participants consumed a verylow caloric diet (654kcal/day, 6 weeks) followed by a low caloric diet (1200–1800 kcal/day, 18weeks). This intervention reduced body weight, blood pressure, and inflammatory markers, such as hsCRP, TNF-α, and nuclear factor kappa β (NFkβ). In addition, as oxidative stress-related parameters, the leukocyte-derived superoxide content, mitochondrial ROS production, and serum protein carbonyl content were decreased in the CR group. Moreover, CR enhanced antioxidant capacities, such as total glutathione and GPx contents, and serum catalase activities in association with the activation of SIRT1 (Fig. 2).15,16 Research on CR in humans has provided valuable information. Moderate nutritional restriction of less than 25% is feasible and safe. Many candidates associated with life span elongation in animal models were conserved and mediated in human CR trials. The suppression of age-related diseases and reductions in cardiovascular risk factors may extend the health span and also life span.

Trials on other modified CR and other modules mimicking CR-induced beneficial signaling Although the beneficial effects of CR on health promotion have been confirmed in humans, strict long-term CR is very hard to accomplish. Hence, alternative approaches

that mimic the beneficial effects of CR are desired. A modified CR protocol, such as intermittent fasting, the restriction of specified nutrient components, and pharmacological supplementation, has been proposed. One alternative method of CR is intermittent fasting. Alternate day fasting (consuming 25% of energy needs on the first day and ad libitum food intake on the following day) for 8 weeks in obese adults decreased body weight and blood pressure and improved lipid profiles in association with reductions in systemic oxidative and inflammatory indicators.51,52 Therefore, an alternative CR regimen with nutritional total caloric reductions will achieve similar beneficial effects to CR. Another modified approach in this research field is specific component restriction. Volek et al. reported that carbon hydrate restriction achieved more favorable improvements in insulin sensitivity and lipid profiles in patients with metabolic syndrome than a low-fat diet.53 A recent review summarized the effectiveness of protein restriction, particularly methionine.54 In animal studies, methionine restriction without CR achieved beneficial results.54 In a human study, a low protein intake reduced overall mortality in 50- to 65-year-old individuals. In contrast, among elderly individuals older than 66 years, mortality in low protein consumers was higher, which may have been due to frailty.55 Interventions that mimic CR without performing CR have been also examined. Among several proposed compounds, resveratrol suppresses inflammation and carcinogenesis, leading to life span elongation and reductions in age-related diseases in many animals.56 In a recent randomized trial on nonobese individuals, a treatment with resveratrol for 30 days increased serum SIRT1 concentrations.57

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Conclusions and future prospective questions The extensive amount of research conducted on CR confirmed life and health span elongation and the retardation of the aging process through multiple mechanisms. The majority of candidate signaling pathways reduce oxidative stress. In human CR studies, common pathways from animal studies were conserved, and reductions in risk factors for age-related diseases were confirmed. However, it has not yet been established whether this intervention is feasible and safe for all individuals because participants enrolled in clinical studies are selected according to strict criteria. Furthermore, the proper level, duration, and age of CR have remained obscure. Therefore, large-scale long-term clinical studies on CR and modified CR regimens are needed in the future.

Applications to other diseases or conditions This review summarizes the beneficial effects of CR on life and health span elongation in animal models and the main proposed signaling pathways for these effects. In human CR, similar changes in longevity-related factors are achieved by CR, and thus CR may delay human aging. Oxidative injury exacerbates not only the aging process but also several cardiovascular diseases, including pressure-overload cardiac hypertrophy and cardiac failure. Diastolic dysfunction in the left ventricle, which is a pathological condition without an effective pharmacological treatment, was attenuated by CR.20,57

Summary points • Aging is an inevitable process in all living organisms, and chronic oxidative stress and inflammation have been implicated in its progression. • Caloric restriction without malnutrition is a powerful nongenetic intervention for the aging process because it reduces susceptibility to age-related diseases and extends the life span of many species. • Multiple signaling pathways contribute to the beneficial effects of CR. In CR, growth and protein synthesis signaling pathways, such as the insulin/ insulin growth factor 1 (IGF-1)-forkhead transcription factor O (FoxO) signaling pathway and target of rapamycin (TOR) pathway, are suppressed. Furthermore, as a metabolic adaptation, AMPdependent protein kinase (AMPK) and sirtuin signaling pathways are activated. • These signaling pathways suppress oxidative injury by reducing the generation of reactive oxygen species (ROS) and promoting the activities of antioxidants.

• Direct evidence for life span elongation by CR in humans is difficult to obtain; however, feasible and continuous CR has been shown to reduce susceptibility to age-related diseases in clinical trials. • Alternative CR regimens achieve similar beneficial effects to CR. Other component restriction regimens and pharmacological interventions that mimic CR, such as resveratrol, exert similar beneficial effects to CR.

References 1. WHO. Global health and ageing; 2011. 2. Fontana L, Partridge L, Longo VD. Extending healthy life span— from yeast to humans. Science 2010;328:321–6. 3. Anton S, Leeuwenburgh C. Fasting or caloric restriction for healthy aging. Exp Gerontol 2013;48:1003–5. 4. Balasubramanian P, Howell PR, Anderson RM. Aging and caloric restriction research: a biological perspective with translational potential. EBioMedicine 2017;21:37–44. 5. Das SK, Balasubramanian P, Weerasekara YK. Nutrition modulation of human aging: the calorie restriction paradigm. Mol Cell Endocrinol 2017;455:148–57. 6. McCay CM, Crowell MF, Maynard LA. The effect of retarded growth upon the length of life span and upon the ultimate body size. 1935. Nutrition 1989;5:155–71. 7. López-Lluch G, Navas P. Calorie restriction as an intervention in ageing. J Physiol 2016;594:2043–60. 8. Colman RJ, Beasley TM, Kemnitz JW, Johnson SC, Weindruch R, Anderson RM. Caloric restriction reduces age. Lated and all-cause mortality in rhesus monkeys. Nat Commun 2014;5:3557. 9. Mattison JA, Roth GS, Beasley TM, Tilmont EM, Handy AM, Herbert RL, Longo DL, Allison DB, Young JE, Bryant M, Barnard D, Ward WF, Qi W, Ingram DK, de Cabo R. Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study. Nature 2012;489:318–21. 10. Mattison JA, Colman RJ, Beasley TM, Allison DB, Kemnitz JW, Roth GS, Ingram DK, Weindruch R, de Cabo R, Anderson RM. Caloric restriction improves health and survival of rhesus monkeys. Nat Commun 2017;8:14063. 11. Merksamer PI, Liu Y, He W, Hirschey MD, Chen D, Verdin E. The sirtuins, oxidative stress and aging: an emerging link. Aging (Albany NY) 2013;5:144–50. 12. Walsh ME, Shi Y, Van Remmen H. The effects of dietary restriction on oxidative stress in rodents. Free Radic Biol Med 2014;66:88–99. 13. Das SK, Balasubramanian P, Weerasekara YK. Nutrition modulation of human aging: the calorie restriction paradigm. Mol Cell Endocrinol 2017;455:148–57. 14. Il’yasova D, Fontana L, Bhapkar M, Pieper CF, Spasojevic I, Redman LM, Das SK, Huffman KM, Kraus WE, CALERIE Study Investigators. Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2-isoprostanes: the CALERIE 2 randomized clinical trial. Aging Cell 2018;17:e12719. 15. López-Domènech S, Martínez-Herrera M, Abad-Jim
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eloën A, Even PC, Cervera P, Le Bouc Y. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature 2003;421:182–7. Mair W, Dillin A. Aging and survival: the genetics of life span extension by dietary restriction. Annu Rev Biochem 2008;77:727–54. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M, Yancopoulos GD, Glass DJ. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 2004;14:395–403. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang TT, Bos JL, Medema RH, Burgering BM. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 2002;419:316–21. Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol 2004;14:885–90. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012;149:274–93. Aris JP, Alvers AL, Ferraiuolo RA, Fishwick LK, Hanvivatpong A, Hu D, Kirlew C, Leonard MT, Losin KJ, Marraffini M, Seo AY, Swanberg V, Westcott JL, Wood MS, Leeuwenburgh C, Dunn Jr. WA. Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast. Exp Gerontol 2013;48:1107–19. Greer EL, Brunet A. Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C. elegans. Aging Cell 2009;8:113–27. Jeon SM, Chandel NS, Hay N. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature 2012;485:661–5. Hayashida S, Arimoto A, Kuramoto Y, Kozako T, Honda S, Shimeno H, Soeda S. Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARalpha in mice. Mol Cell Biochem 2010;339:285–92. Imai S, Guarente L. Ten years of NAD-dependent SIR2 family deacetylases: implications for metabolic diseases. Trends Pharmacol Sci 2010;31:212–20.

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34. Luo H, Chiang HH, Louw M, Susanto A, Chen D. Nutrient sensing and the oxidative stress response. Endocrinol Metab 2017;28:449–60. 35. Qiu X, Brown K, Hirschey MD, Verdin E, Chen D. Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation. Cell Metab 2010;12:662–7. 36. Someya S, Yu W, Hallows WC, Xu J, Vann JM, Leeuwenburgh C, Tanokura M, Denu JM, Prolla TA. Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction. Cell 2010;143:802–12. 37. Short KR, Bigelow ML, Kahl J, Singh R, Coenen-Schimke J, Raghavakaimal S, Nair KS. Decline in skeletal muscle mitochondrial function with aging in humans. Proc Natl Acad Sci U S A 2005;102:5618–23. 38. Yu BP. Membrane alteration as a basis of aging and the protective effects of calorie restriction. Mech Ageing Dev 2005;126:1003–10. 39. López-Domínguez JA, Ramsey JJ, Tran D, Imai DM, Koehne A, Laing ST, Griffey SM, Kim K, Taylor SL, Hagopian K, Villalba JM, López-Lluch G, Navas P, McDonald RB. The influence of dietary fat source on life span in calorie restricted mice. J Gerontol A Biol Sci Med Sci 2015;70:1181–8. 40. Strom A, Jensen RA. Mortality from circulatory diseases in Norway 1940-1945. Lancet 1951;1:126–9. 41. Meydani M, Das S, Band M, Epstein S, Roberts S. The effect of caloric restriction and glycemic load on measures of oxidative stress and antioxidants in humans: results from the CALERIE trial of human caloric restriction. J Nutr Health Aging 2011;15:456–60. 42. Weiss EP, Racette SB, Villareal DT, Fontana L, Steger-May K, Schechtman KB, Klein S, Holloszy JO, Washington University School of Medicine CALERIE Group. Improvements in glucose tolerance and insulin action induced by increasing energy expenditure or decreasing energy intake: a randomized controlled trial. Am J Clin Nutr 2006;84:1033–42. 43. Fontana L, Villareal DT, Weiss EP, Racette SB, Steger-May K, Klein S, Holloszy JO, Washington University School of Medicine CALERIE Group. Calorie restriction or exercise: effects on coronary heart disease risk factors. A randomized, controlled trial. Am J Physiol Endocrinol Metab 2007;293:E197–202. 44. Hofer T, Fontana L, Anton SD, Weiss EP, Villareal D, Malayappan B, Leeuwenburgh C. Long-term effects of caloric restriction or exercise on DNA and RNA oxidation levels in white blood cells and urine in humans. Rejuvenation Res 2008;11:793–9. 45. Civitarese AE, Carling S, Heilbronn LK, Hulver MH, Ukropcova B, Deutsch WA, Smith SR, Ravussin E, CALERIE Pennington Team. Calorie restriction increases muscle mitochondrial biogenesis in healthy humans. PLoS Med 2007;4:e76. 46. Ravussin E, Redman LM, Rochon J, Das SK, Fontana L, Kraus WE, Romashkan S, Williamson DA, Meydani SN, Villareal DT, Smith SR, Stein RI, Scott TM, Stewart TM, Saltzman E, Klein S, Bhapkar M, Martin CK, Gilhooly CH, Holloszy JO, Hadley EC, Roberts SB, CALERIE Study Group. A 2-year randomized controlled trial of human caloric restriction: feasibility and effects on predictors of health span and longevity. J Gerontol A Biol Sci Med Sci 2015;70:1097–104. 47. Fontana L, Villareal DT, Das SK, Smith SR, Meydani SN, Pittas AG, Klein S, Bhapkar M, Rochon J, Ravussin E, Holloszy JO, CALERIE Study Group. Effects of 2-year calorie restriction on circulating levels of IGF-1, IGF-binding proteins and cortisol in nonobese men and women: a randomized clinical trial. Aging Cell 2016;15:22–7. 48. Sparks LM, Redman LM, Conley KE, Harper ME, Yi F, Hodges A, Eroshkin A, Costford SR, Gabriel ME, Shook C, Cornnell HH, Ravussin E, Smith SR. Effects of 12 months of caloric restriction on muscle mitochondrial function in healthy individuals. J Clin Endocrinol Metab 2017;102:111–21.

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49. Li C, Feng F, Xiong X, Li R, Chen N. Exercise coupled with dietary restriction reduces oxidative stress in male adolescents with obesity. Sports Sci 2017;35:663–8. 50. Buchowski MS, Hongu N, Acra S, Wang L, Warolin J, Roberts 2nd. LJ. Effect of modest caloric restriction on oxidative stress in women, a randomized trial. PLoS One 2012;7:e47079. 51. Varady KA, Bhutani S, Church EC, Klempel MC. Short-term modified alternate-day fasting: a novel dietary strategy for weight loss and cardioprotection in obese adults. Am J Clin Nutr 2009;90:1138–43. 52. Johnson JB, Summer W, Cutler RG, Martin B, Hyun DH, Dixit VD, Pearson M, Nassar M, Telljohann R, Maudsley S, Carlson O, John S, Laub DR, Mattson MP. Alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma. Free Radic Biol Med 2007;42:665–74. 53. Volek JS, Phinney SD, Forsythe CE, Quann EE, Wood RJ, Puglisi MJ, Kraemer WJ, Bibus DM, Fernandez ML, Feinman RD. Carbohydrate restriction has a more favorable impact on the metabolic syndrome than a low fat diet. Lipids 2009;244:297–309. 54. Kitada M, Ogura Y, Monno I, Koya D. The impact of dietary protein intake on longevity and metabolic health. EBioMedicine 2019; 43:632–40.

55. Levine ME, Suarez JA, Brandhorst S, Balasubramanian P, Cheng CW, Madia F, Fontana L, Mirisola MG, GuevaraAguirre J, Wan J, Passarino G, Kennedy BK, Wei M, Cohen P, Crimmins EM, Longo VD. Low protein intake is associated with a major reduction in IGF-1, cancer, and overall mortality in the 65 and younger but not older population. Cell Metab 2014; 19:407–17. 56. Bhullar KS, Hubbard BP. Lifespan and healthspan extension by resveratrol. Biochim Biophys Acta 2015;1852:1209–18. 57. Mansur AP, Roggerio A, Goes MFS, Avakian SD, Leal DP, Maranhão RC, Strunz CMC. Serum concentrations and gene expression of sirtuin 1 in healthy and slightly overweight subjects after caloric restriction or resveratrol supplementation: a randomized trial. Int J Cardiol 2017;227:788–94.

Further reading Meyer TE, Kovács SJ, Ehsani AA, Klein S, Holloszy JO, Fontana L. Longterm caloric restriction ameliorates the decline in diastolic function in humans. J Am Coll Cardiol 2006;47:398–402.

1. Oxidative stress and aging

C H A P T E R

3 Cardiovascular disease in aging and the role of oxidative stress Lucie Orliagueta, Vinood B. Patelb, Victor R. Preedyc, Fawaz Alzaida a

b

Immunity and Metabolism of Diabetes Team, INSERM Unit 1138, Cordeliers Research Centre, Paris, France Department of Biomedical Sciences, School of Life Sciences, University of Westminster, London, United Kingdom c Diabetes and Nutritional Sciences Research Division, School of Medicine, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom

siRNA SIRT TSC1 TSC2 UCP2 VEGF

List of abbreviations 3-NPA ADP AKT AMP AMPK AP-1 ATP CAT ERK FADH FOX FOXO GPx GST HIF-1 IGF1 LDLs LKB1 mCLK1 MiR1 miRNA Mn SOD mTOR mTORC1 mTORC2 NADH NADPH p66Shc PI3K PIT1 PKCB PROP1 PTP RNA ROS SIR2

3-nitropropionic acid adenosine diphosphate protein kinase B adenosine monophosphate adenosine monophosphate-activated protein kinase activator protein 1 adenosine triphosphate catalase extracellular signal-regulated kinase flavin adenine dinucleotide forkhead box O class forkhead box glutathione peroxidase glutathione S-transferase hypoxia-inducible factor 1 insulin-like growth factor 1 low-density lipoproteins liver kinase B1 mammalian CLOCK1 microRNA1 microribonucleic acid manganese superoxide dismutase mammalian target of rapamycin mammalian target of rapamycin complex 1 mammalian target of rapamycin complex 2 nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate 66 kDa splice variant of the Shc locus phosphoinositide 3-kinase pituitary transcription factor 1 protein kinase C-β homeobox protein prophet of pituitary transcription factor 1 permeability transition pore ribonucleic acid reactive oxygen species sirtuin

Aging https://doi.org/10.1016/B978-0-12-818698-5.00003-1

small interfering ribonucleic acid sirtuin family member tuberous sclerosis complex 1 tuberous sclerosis complex 2 mitochondrial uncoupling protein 2 vascular endothelial growth factor

Introduction Aging is an inevitable fate for any organism, and unfortunately the deterioration that occurs with aging predisposes individuals to numerous conditions. Cardiovascular diseases represent a large group of conditions with a dramatically increased occurrence with age. As well as the emotive impact on the family unit and communities, cardiovascular diseases claim a large number of lives and cause a significant detriment to the economy through healthcare costs and productivity losses. In Europe, this amounts to 1.9 million deaths and a cost of €196 billion per year.1 One-third of all deaths in the United States are caused by cardiovascular disorders, with more than 83 million Americans currently living with one or more of these fatal conditions. Furthermore, cardiovascular disorders cost the American economy $444 billion a year, equating to one-sixth of the country’s spending on health.2 Biological aging predisposes individuals to cardiovascular disease through age-related perturbation of systemic and/or cellular oxidative balance, that is, discord between the rates of generation and clearance of reactive oxygen species (ROS). Due to this increased susceptibility, death related to cardiovascular events rapidly becomes more common with aging. For example, in the United Kingdom in 2009, there were approximately 180,000 deaths due to

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Copyright © 2020 Elsevier Inc. All rights reserved.

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3. Oxidative stress and cardiovascular disease

cardiovascular disease, 72% of which were in populations over the age of 753 (Figs. 1 and 2). Oxidative stress has long been known to increase with the aging process, and it independently causes a greater risk of cardiovascular disease. Biologic systems are equipped to neutralize endogenous oxidative stress and respond appropriately to oxidative challenges. Throughout biologic aging, these protective mechanisms decline,

and thus the oxidative theory of aging extends to pathophysiological developments that predispose individuals to a much higher risk of conditions such as cardiovascular disease. The cardiovascular system is particularly sensitive to endogenous oxidative stress as the myocardium is particularly rich in mitochondria and mitochondrial metabolism is the main source of cellular-free radical generation.4

FIG. 1 Rates of death from cardiovascular disease increase with age in the United Kingdom. Cardiovascular diseases represent the most common causes of death in the United Kingdom. This figure shows the deaths in the United Kingdom in 2009 that are due to cardiovascular disease. The risk of death from cardiovascular disease increases with age, with the highest risk group being those above 75 years of age; this trend is in both men and women. Data from Nichols M, Townsend N, Luengo-Fernandez R, et al. European cardiovascular disease statistics 2012. Brussels/Sophia Antipolis: European Heart Network/European Society of Cardiology; 2012.

FIG. 2

Impact of cardiovascular disease in Europe. Top chart shows the leading causes of death of men in Europe in 2012; cardiovascular disease, stroke, and coronary heart disease are the leading causes of death. Bottom chart shows the leading causes of death of women in Europe in 2012; cardiovascular disease, stroke, and coronary heart disease cause more than half of the deaths in Europe. CVD, cardiovascular disease; CHD, coronary heart disease. Data from Scarborough P, Wickramasinghe K, Bhatnagar P, Rayner M. Trends in coronary heart disease 1961–2011. London: British Heart Foundation; 2011.

1. Oxidative stress and aging

Physiology of the aging cardiovascular system

The genes that regulate cardiovascular physiologic function and responses to oxidative challenge in aging have been identified as longevity genes, a subset of which interacts extensively as the longevity network.5 Biologic aging is also heavily attributed to the telomere theory. The telomere theory states that telomere shortening, which is accelerated by oxidative stress, is responsible for much of the age-related deterioration.6 The implication of oxidative stress in age-related physiologic decline is well established and supported by data showing a marked increase in plasma potential for oxidative damage with age (Fig. 3).7 This increased oxidant potential is indicative of the imbalance between the generation and clearance of ROS throughout aging, contributing to the pathogenesis of age-related cardiovascular disease. Furthermore, as age increases, the antioxidant activity of several antioxidant enzymes in the cardiovascular system decreases (Fig. 4A). The oxidative balance of the cardiovascular system is further disturbed by the increased oxidative stress from mitochondria and lipid peroxidation (Fig. 4B). In this chapter, we describe the physiologic changes that accompany aging and that predispose individuals to a decline in vascular function and the risk of cardiovascular diseases. We focus specifically on the effects of oxidative stress and the dual role it plays in aging and in the pathogenesis of cardiovascular disease.

Physiology of the aging cardiovascular system The cardiovascular system is the most widely reaching system in the human body, delivering oxygenated blood and nutrients to all tissues. However, like in any system,

21

aging alone will cause a physiologic and functional decline that increases susceptibility to complications. In the cardiovascular system the decline in functional capacity greatly increases the risk of hypertension and atherosclerosis, leading to life-threatening events such as myocardial infarction or stroke. Aging and the associated functional decline are greatly accelerated by systemic and local oxidative stress. Fig. 5 summarizes the physiologic changes that occur in an aging cardiovascular system. These effects mainly cause a decline in endothelial function, left ventricle systolic reverse capacity, and left ventricular diastolic function.5 Interestingly the effects of aging in either the vascular or cardiac system will induce a compensatory and potentially pathologic change in the other. For example, increased arterial stiffness causes accumulation of fibrotic tissues or hypertrophy in the myocardium.5 The main functional changes that occur with aging are a decline in heart rate and a decline in cardiac output. The decline in heart rate is due to cell loss in the sinoatrial node and impedance of electrical impulses from structural changes such as hypertrophy and fibrosis. These changes are due to an age-related decline in stress response mechanisms.5, 8 The decline in cardiac output begins with hemodynamics adapting to meet changing requirements with age. The cardiovascular system’s immediate response stimulates myocardial hypertrophy that increases cardiac output accordingly. These adaptations subsequently lead to a decline in cardiac output and overall cardiovascular function. Myocardial hypertrophy, independently or as part of age-related compensation, is a risk factor for morbidity and mortality associated with cardiovascular disease.5, 8

FIG. 3 The potential for oxidative damage increases with age. Oxidative stress contributes to the biologic aging process and is a central part of the pathogenesis of cardiovascular disease. This figure shows the clear relationship between aging and the increased potential for oxidative stress (plasma oxidant potential). An increased potential for oxidative stress increases the risk of developing age-related pathologies, including cardiovascular disease. Reproduced with permission from Mehdi MM, Rizvi SI. N,N-dimethyl-p-phenylenediamine dihydrochloride-based method for the measurement of plasma oxidative capacity during human aging. Anal Biochem 2013;436:165–7. https://doi.org/10.1016/j.ab. 2013.01.032.

1. Oxidative stress and aging

FIG. 4

Decrease in antioxidant enzyme activity and increase in oxidative stress in the aging cardiovascular system. There is decreased activity of several antioxidant enzymes, namely, manganese superoxide dismutase (Mn SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST). (A) This shows the percentage decrease in activity of these enzymes between young and aged mouse hearts. As well as the decrease in these antioxidant enzymes, there is increased oxidative stress that occurs through increased lipid peroxidation and the generation of mitochondrial ROS. (B) This shows the increased production of ROS in hearts from aged versus young mice. Mn SOD, manganese superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferase; MDA, malondialdehyde; RFU, relative fluorescence units; ROS, reactive oxygen species. Data from Sudheesh NP, Ajith TA, Ramnath V, Janardhanan KK. Therapeutic potential of Ganoderma lucidum (Fr.) P. Karst. against the declined antioxidant status in the mitochondria of post-mitotic tissues of aged mice. Clin Nutr 2010;29:406–12. https://doi.org/10.1016/j.clnu. 2009.12.003.ART: Please replace with attached figure.

FIG. 5

Age-related changes in the cardiovascular system. In the heart, several functional and structural changes occur with the aging process. Panel (A): Young heart with normal thickness of the myocardium and no compromised function. Panel (B): Young artery with normal sized lumen and vessel wall; adaptive contractility and relaxation is maintained. Panel (C): Aged heart with myocardial hypertrophy; cardiomyocytes are undergoing senescence and thus have compromised function. Hypertrophy and fibrotic tissues will lead to impeded signaling from the sinoatrial node. Panel (D): Aged artery with narrower lumen, increased wall thickness; adaptive contractility and relaxation is lost. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle. Reproduced with permission from Shaik S, Wang Z, Inuzuka H, Wei W. In: Z. Wang, editor. Endothelium aging and vascular diseases, senescence and senescence-related disorders. InTech; 2013. https://doi.org/10.5772/53065. Available from: http://www.intechopen.com/books/senescence-andsenescence-related-disorders/endothelium-agingand-vascular-diseases.

Young

Aged

LA RA

RV

LV

Hypertrophy

Normal

(A)

(C) Cardiomyocytes

Cardiomyocytes

(B)

Senescent cells

(D)

The molecular basis of oxidative stress as applied to the cardiovascular system

The molecular basis of oxidative stress as applied to the cardiovascular system In any cell a multitude of reactions require the transfer of electrons. Whenever electrons are exchanged, there is a change in oxidative state of the molecules involved in these reactions. Oxidation or reduction constitutes the gain or loss of electrons, respectively. These processes occur simultaneously and are termed redox reactions, where a reductant will be oxidized by donating an electron to an oxidant.9 Reactive oxygen species (ROS), generated as by-products of the aforementioned redox reactions, contain unpaired electrons, making them highly reactive and dangerous sources of oxidative stress. The main endogenous process that generates ROS is mitochondrial oxidative phosphorylation.9 These mitochondrial reactions are particularly relevant to the cardiovascular system, as 45% of the heart’s cellular volume is taken up by mitochondria.4 During oxidative phosphorylation, electrons from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH) are processed via four mitochondrial enzyme complexes. The end product of this pathway is the production of adenosine triphosphate (ATP). Harmful ROS are generated when electrons lost from mitochondrial complexes I and II form superoxide radicals. Furthermore, NADH and FADH are free to react with other redox compounds throughout the mitochondrial pathway.9 As vessels age, they produce more ROS, leading to functional impairment. Increased production of the superoxide anion leads to increased reaction of this ROS with nitric oxide. This reaction will not only inhibit the biological activity of nitric oxide as a vasodilator but also results in the formation of peroxynitrite. Peroxynitrite is a powerful membrane-permeable oxidant that inactivates several enzymes, including free radical scavengers, through substrate nitration. Age-dependent increases in nitric oxide synthase expression are associated with the peroxynitrite formation and are therefore implicated in age-related oxidative damage to vasculature.9, 10 Age-related oxidative stress is also central to pathogeneses of atherosclerosis, a starting point of more serious cardiovascular conditions. Endothelial cells and vascular smooth muscle cells produce reactive oxygen and nitrogen species that oxidize low-density lipoproteins (LDL). Oxidized LDL enter subendothelial spaces where they initiate atherosclerosis. The oxidation process will increase mitochondrial rupture and the release of proapoptotic molecules to the cytosol, increasing plaque cell apoptosis. Furthermore the scavenging process for reactive nitrogen species increases endothelial dysfunction, smooth muscle cell proliferation, leukocyte adhesion, and inflammatory responses. Thus the generation and clearance of oxidative stress in vasculature is an extremely significant regulator of age-related cardiovascular decline.5, 9

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Alongside gross cardiovascular system physiologic adaptation, molecular and genetic mechanisms are known to mediate cardiovascular responses to oxidative stress and age-related decline. The three leading molecular mediators are the involvement of longevity genes and the longevity network (which are discussed later) and the telomere theory of aging that is also strongly associated with cardiovascular decline.5, 6 Telomeres are structures of repeating nucleotides at the end of eukaryotic chromatids that are shortened during replication. This mechanism exists to protect valuable genetic data from being lost by incomplete replication. Telomere length and thus protective capacity are determined genetically, are decreased by oxidative stress, and vary with life span.6 In conditions such as cardiovascular disease, with a significant multifactorial etiology involving both aging and metabolic oxidative stress, it is important to consider the role of telomere attrition. This is because the telomere theory is a candidate for distinguishing individual variability of risk and susceptibility, a factor that remains largely unknown, unlike population variability, which is attributed to known factors such as smoking or ethnicity. When shortened to a critical length, telomeres will drive senescence signaling mechanisms. Dysregulation in the pathways of senescence is a strong underlying mechanism leading to cardiovascular decline. There have also been strong associations between systemic cell typespecific telomere shortening and the development of cardiovascular disease. For example, the presence of shortened telomeres in circulating leukocytes is associated with the development of coronary artery disease. In observational studies the presence of shortened telomeres precedes the development of clinically relevant disease, indicating a more causal role rather than a consequential effect of cardiovascular decline.11, 12 Many interesting studies have investigated this hypothesis. For example, Cawthon et al.13 found that in subjects aged over 60, a shorter telomere length was strongly associated with a high cardiac mortality rate within a decade. In this study the presence of other classic cardiovascular risk factors did not explain the higher mortality rate. Furthermore, as telomeres shorten with aging, the average loss of telomeres can be equated to the number of years. Those with coronary artery disease are, on average, 8–12 years older, in terms of telomere loss. Telomere attrition has been specifically associated with a number of cardiovascular pathologic events, including congestive heart failure, peripheral vascular disease, and carotid artery atherosclerosis.6, 11–13 There are continuously strengthening links between aging, cardiovascular disease, and telomere shortening. Oxidative stress is implicated in negatively affecting all three processes: accelerating telomere loss, accelerating aging, and a significant pathologic component of cardiovascular disease.6 Whether a common causal pathway is

1. Oxidative stress and aging

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3. Oxidative stress and cardiovascular disease

TABLE 1 Longevity genes.a Gene symbol

Gene name

Sequence IDs

SIRT1

Sirtuin 1

NM_001142498.1; NM_012238.4

SIRT3

Sirtuin 3

NM_00107524.2; NM_012239.5

SIRT7

Sirtuin 7

NM_016538.2

IGF1

Insulin-like growth factor 1

NM_000618.3; NM_001111283.1; NM_001111284.1; NM_00111285.1;

FOXO1

Forkhead box O1

NM_002015.3

FOXO3

Forkhead box O3

NM_001455.3; NM_201559.2

PIT1

Pituitary-specific positive class 1 homeobox

NM_000306.2; NM_001122757.1

BUBR1

BUB1 mitotic checkpoint serine/threonine kinase

NM_001278616; NM_001278617.1; NM_004336.4

AMPKa

Protein kinase, AMPactivated, alpha 1 catalytic subunit

NM_006251.1; NM_206907.3

mTORa

Mechanistic target of rapamycin

NM_004958.3

p66shc

Src homology 2 domaincontaining transforming protein C1

NM_001113331.2; NM_011368.5

KL

Klotho

NM_004795

CAT

Catalase

NM_001752

CLK1

Circadian locomotor output cycles kaput

NM_001254772

a

a

FIG. 6 Factors that lead to telomere attrition and a higher risk of cardiovascular disease. The factors that affect telomere length help to explain interindividual variation in the risk of developing cardiovascular disease. Telomere length will vary genetically at birth and will shorten with age and exposure to oxidative stress (e.g., smoking or metabolic conditions such as obesity). Cell replication also shortens telomere length; when a critical length has been reached telomeres activate senescence signaling that, in turn, increases the risk of cardiovascular disease. Modified from Samani NJ, van der Harst P. Biological ageing and cardiovascular disease. Heart 2008;94:537–9. https://doi.org/10.1136/hrt. 2007.136010.

responsible for the cardiovascular aging phenotype, for telomere attrition, and for sensitivity to oxidative stress is an extremely exciting area of current research. Fig. 6 summarizes the factors that increase telomere attrition, senescence signaling, and, subsequently, a higher risk of cardiovascular disease.

Longevity genes and the longevity network Longevity genes regulate oxidative stress response pathways and biologic aging. These factors are major parts of cardiovascular disease pathology.5 The list of genes includes the sirtuins (SIR2), insulin-like growth factor-1 (IGF1), CLOCK1 (mCLK1), adenosine monophosphateactivated protein kinases (AMPK), p66Shc, catalase, and klotho; see Table 1 for a complete list of the genes discussed in the following section. A subset of the longevity genes interacts extensively and constitutes the longevity network, the functions, and interactions of which are addressed first.

Genes of the longevity network: SIR2, IGF-1, AMPK, and mTOR The sirtuins Sirtuins (SIR2) are a class of evolutionarily conserved enzymes. In mammals, there are seven members of the SIR2 family with diverse cellular localizations. These proteins have a wide range of functions, namely, ribosyltransferase and deacetylase activity, and functions in aging, transcription, apoptosis, inflammation, DNA

a

These genes have been found to have a substantial link with age-related physiologic decline in cardiovascular function and with cellular and systemic responses to oxidative stress. A number of these genes (SIRT1, AMPK, IGF-1, and mTOR) form the longevity network, a network that regulates biologic aging and therefore the development of cardiovascular disease.

damage and repair, cell cycle regulation, stress resistance, and mitochondrial function.14

The cardiovascular system and SIR2 proteins SIR2 family members 1, 3, and 7 (SIRT1, SIRT3, and SIRT7, respectively) have proven roles in the cardiovascular system. Cardiac-specific overexpression of SIRT1 in mice delays age-dependent cardiomyopathy and decreases stress-induced apoptosis. Knockout and constitutive overexpression is detrimental, increasing ischemia-reperfusion injury, cardiomyopathy, apoptosis, and oxidative stress. SIRT1 also effects vasculature, inhibiting angiotensin II-mediated vascular smooth muscle hypertrophy. It regulates angiogenesis and prevents arterial calcification and

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stiffness.15–17 Knockout of mitochondrial SIRT3 and nuclear SIRT7 causes age-dependent, pressure-induced cardiac hypertrophy, and inflammatory cardiomyopathy. SIRT3 and SIRT7 protect from oxidative damage and prevent cardiovascular remodeling and inflammation.18, 19

induction of IGF1 delays age-related degeneration. Some studies report opposite effects in mammals. This difference is possibly explained by the complex autocrine and paracrine signaling pathways that are targeted by IGF1 and are not present in simpler organisms.5, 22

Oxidative stress and SIR2 proteins

Oxidative stress and IGF1

The roles of SIR2 proteins in oxidative stress are complex and are member, site, and species specific. SIRT1, SIRT6, and SIRT7 proteins have been strongly associated with mediating oxidative stress responses in mammals. Expression of SIRT1 ameliorates the aggravated oxidative stress seen in cardiovascular disease.20 SIRT6 is expressed in a number of tissues, including the heart; its overexpression promotes resistance to oxidative stress and associated DNA damage. SIRT6 substrate specificity is very high, exerting deacetylase activity to maintain chromatin function, in turn promoting telomere stability. Therefore SIRT6 prevents cell senescence through mediating resistance to oxidative stress, ameliorating DNA damage and preserving telomere length.21 As for SIRT7, diminished expression in cardiomyocytes decreases oxidative stress resistance and increases apoptosis by up to 200%. This is mediated via hyperacetylation of the p53 protein and has been confirmed in haplotype mice with diminished SIRT7 expression.19

Oxidative stress is associated with decreased plasma IGF1 in humans, leading to cardiac and mitochondrial dysfunction. When mice have cardiac-specific overexpression or exogenous administration of IGF1 under conditions that induce cardiovascular disease, apoptosis and mitochondrial dysfunction are decreased alongside lower systemic and myocardial oxidative stress. Furthermore, IGF1 promotes protective regulation of other mediators of aging and cardiovascular risk (e.g., FOXO and mTOR). Despite protective effects of IGF1 on cardiovascular function, remodeling and hypertrophy still occur due to preserved mitochondrial function and increased cell survival under prooxidant conditions.25, 26 The interaction between IGF1 and its receptor regulates cell growth, transformation, and survival under oxidative stress. When this interaction is perturbed (e.g., in haplotype mice and in myoblasts cultured from them), there is increased resistance to oxidative stress.27 Studies in other types of cell show opposite effects; for example, increased IGF1-signaling confers protection from oxidative stress in microglial cells and in induced pluripotent stem cells.28 Studies applying small interfering RNA (siRNA) have shown the complexity of these mechanisms. The effects of perturbing these mechanisms will vary upstream or downstream of IGF1 signaling.28 Through the aforementioned studies, microRNA-1 (MiR1) targeting of the 30 -UTR region of the IGF1 gene has been shown to regulate the cytoprotective properties and prevent oxidative stress-induced apoptosis signaling.28

Insulin-like growth factor 1 One of the initial genes to be identified as a longevity gene, insulin-like growth factor 1 (IGF1), encodes a peptide structurally similar to insulin with an important function in early growth and an anabolic effect in adults. IGF1 exerts its effects by binding to its receptor that is expressed on many cell types and tissues. From this binding site, IGF1 inhibits apoptosis and acts as a stimulator of cell growth and proliferation.5, 22 In mammals, the loss of this protein’s function causes dwarfism, deficient growth, or postnatal death from respiratory failure.23

The cardiovascular system and IGF1 Several studies have implicated IGF1 signaling in the maintenance of cardiovascular health. In mice, cardiacspecific overexpression of IGF1 prevents cell death after myocardial infarction and reduces hypertrophy, ventricular dilation, and diabetic cardiomyopathy. However, other overexpression studies have shown negative effects, including hypertrophy and diminished recovery after ischemia. Live knockout of mammalian IGF1 increases cardiomyocyte cell death under oxidative stress.24–26 In simple organisms, with a primitive cardiovascular system, the

Forkhead box transcription factors Forkhead box (FOX) proteins are a large family of transcription factors regulating expression of genes that control cell growth, proliferation, differentiation, and aging. Of particular interest to cardiovascular health and aging are the O class FOX proteins (FOXO) that also regulate metabolism and stress tolerance. They are posttranslationally controlled by ubiquitination, phosphorylation, and acetylation.29

The cardiovascular system and FOXO proteins Maintaining finely tuned function of FOXO proteins is necessary for life. Deletion of the FOXO1 member is

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embryonically lethal and causes deficient cardiac and vascular growth. Interestingly, cardiac-specific overexpression of FOXO1 is also lethal and causes reduced heart size and myocardium thickness as well as heart failure and impaired cardiomyocyte proliferation.29, 30 A variant of FOXO3 is commonly found in centenarians and is thought to delay aging and preserve cardiovascular health. Overexpression of the FOXO3a variant decreases cardiomyocyte size, whereas deficiency increases endothelial nitric oxide synthase expression and promotes postnatal angiogenesis and vessel formation.31, 32

Oxidative stress and FOXO proteins FOXO signaling maintains homeostasis and mediates responses to environmental pathologic changes, such as oxidative stress. FOXO3a is inhibited by the protein kinase B (AKT) pathway and upregulated by hypoxic conditions. FOXO3a is known to inhibit MYC-mediated adaptive mitochondrial metabolism. Under cellular oxidative stress, AKT is inhibited, which increases FOXO3a signaling. FOXO3a, via inhibition of MYC signaling, prevents increased mitochondrial metabolism under hypoxia. In this cascade the knockdown of FOXO3a allows MYC signaling to increase mitochondrial oxidative stress generation.32, 33 Furthermore, under metabolic oxidative stress, FOXO3a signaling disrupts vascular function by facilitating degradation of vascular calcium/potassium channels. In mice, suppression of FOXO3a preserves cardiovascular function under metabolic oxidative stress.34

Adenosine monophosphate-activated protein kinase Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is involved in cellular energy homeostasis, glucose metabolism, lipid metabolism, cell growth, polarity, gene expression, and autophagy. AMPK regulates cellular energy metabolism by mitophagy and mitogenesis: destruction of defective mitochondria and activation of mitochondrial biogenesis, respectively. AMPK is also a metabolic energy sensor for the AMP/ADP ratio, stimulating ketogenesis and hepatic fatty acid oxidation and inhibiting lipogenesis, triglyceride, and cholesterol synthesis.35

ameliorating damage from ischemia-reperfusion injury and preventing pressure-overload hypertrophy.36, 37

Oxidative stress and AMPK AMPK is a target of pharmaceutical regulation for its role in defense against mitochondrial oxidative stress. Endothelial mitochondrial ROS are implicated in agerelated cardiovascular decline. Endothelial oxidative stress and AMPK expression increase during impaired vasodilation and hypertension from coronary artery disease and diabetes. Diabetes is strongly associated with systemic oxidative stress and is a leading comorbidity with aging cardiovascular disease.38 Further observations of mitochondrial oxidative stress and AMPK regulation have been made in human saphenous vein endothelial cells. When these cells, under oxidative stress, are incubated with a mitochondrialtargeted antioxidant, AMPK expression decreases, providing evidence for the role of AMPK in mitochondrial oxidative stress defense.39 It is necessary to maintain the delicate balance in production and clearance of such reactive species given their functions as signaling molecules in normal cardiovascular function and adaptability. Cardiovascular dysfunction occurs when their rate of production overtakes endogenous free radical scavenging processes. In this regard, AMPK’s role as an energy sensor is clear as the major sources of oxidative stress are mitochondrial nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. There is currently strong evidence to support AMPK’s role as a suppressor of NADPH oxidase activity and therefore reactive species generation.38, 39 AMPK also has a role in regulating adaptive angiogenesis under oxidative stress. This has been shown by rotenone treatment in coronary artery endothelial cells (rotenone increases oxidative stress and inhibits mitochondrial complex I). Vascular tube formation induced by vascular endothelial growth factor (VEGF) is inhibited by rotenone treatment. In the same process, the rotenone induction of oxidative stress increases expression of AMPK. Interestingly, knockdown of AMPK in rotenone treatment preserves VEGF-induced tube formation.39

Mammalian target of rapamycin (mTOR) The cardiovascular system and AMPK In humans, hereditary syndromes due to AMPK mutations have a pathologic component in cardiovascular decline. AMPK mutations cause hypertrophic cardiomyopathy and ventricular preexcitation. Diminished expression of AMPK exacerbates the effects of myocardial infarction, whereas overexpression is cardioprotective,

Rapamycin is an antifungal compound originally discovered in soil samples from Easter Island. It is clinically used as an immunosuppressant after organ transplantation.40 Rapamycin inhibits the activity of the mammalian target of rapamycin (mTOR). The product of mTOR is a serine/threonine protein kinase that also integrates products of upstream pathways, including insulin and IGF1. These functions are mediated via two complexes

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Other genes and pathways in oxidative stress and age-related cardiovascular disease

(mTORC1 and mTORC2). The mTOR pathway is dysregulated in a number of human metabolic pathologies that induce oxidative stress, such as diabetes and obesity.6

The cardiovascular system and mTOR Cardiac inhibition of mTOR activity reverses pressureoverload hypertrophy, via inhibition of mTOR’s control over cell size and protein translation.41 The mTOR protein also interacts with AKT and phosphatidylinositide 3-kinase (PI3K) in the PI3K/AKT/mTOR pathway, which mediates hypoxia-induced angiogenesis. Rapamycin perturbation of the PI3K/AKT/mTOR pathway inhibits vessel growth, normally mediated by hypoxiainducible factor 1 (HIF1) and VEGF.42 The PI3K/AKT/ mTOR pathway is central to many other signaling pathways, making these genes and their products vital to the understanding of oxidative damage, cardiovascular health, and aging.

Oxidative stress and mTOR Oxidative stress influences target upstream and downstream of the mTOR pathway. Sustained increased activity of the mTOR pathway is a strong causal candidate for cardiovascular decline with age and oxidative stress. Furthermore, mTOR’s response to oxidative stress is associated with the inflammatory pathways that progress cardiovascular disease.43 In human coronary artery endothelial cells, inducing oxidative stress by rotenone inhibits mTOR activity.39 Furthermore, mTOR regulates arterial responses to oxidative stress alongside AMPK. Under oxidative stress, arterial contraction and compliance are compromised. When rapamycin is applied to mouse tissues, arterial contraction is preserved, indicating that mTOR mediates adaptive loss of contractile function under oxidative stress.44 The effect of mTOR on cardiac remodeling is associated with and possibly governed by the body’s renin-angiotensin-aldosterone axis, which is central to the oxidative stress response, maintaining blood pressure and maintaining arterial vasoconstriction. When renin is overexpressed in rats, there is a concomitant increase in mTOR activity and metabolic, biochemical, and physical manifestations of oxidative stress and of cardiovascular system decline.45

The longevity network: SIR2, IGF1, AMPK, and mTOR The longevity network comprises the interactions between SIRT1, IGF1, mTOR, and AMPK (see Fig. 7).5 SIRT1 is cardioprotective and increases stress resistance when mildly overexpressed. It regulates the hepatic AMPK

27

pathway via the upstream liver kinase B1 (LKB1). IGF1 is regulated both directly by SIRT1 and via the mitochondrial uncoupling protein 2 (UCP2). SIRT1 also inhibits the mTOR pathway through the complexes formed by tuberous sclerosis 1 and 2 (TSC1 and TSC2).46 IGF1 interacts with both mTOR and SIRT1 pathways. FOXO is a downstream effector of SIRT1’s action on IGF1.5 The mTOR pathway is stimulated by AKT signaling via IGF1, and it is inhibited by AMPK and phosphorylation of the TSC1 and TSC2 complex.47 AMPK, in turn, activates SIRT1 and IGF1 signaling. SIRT1 activity increases with levels of nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide adenine dinucleotide (NAD). In contrast, IGF1 is stimulated through the extracellular signal-regulated kinase cascade (ERK1/2).5, 47 The longevity network has strong links to the oxidative stress response and aims to preserve both cellular and organ function throughout aging. In the longevity network mitochondrial function, metabolism and protein synthesis are modulated to preserve cellular function, whereas stress resistance and autophagy are stimulated to promote cellular senescence or proliferation to preserve organ function above cell fate.5, 46, 47

Other genes and pathways in oxidative stress and age-related cardiovascular disease CLOCK1 CLOCK1 (mCLK1) is a mitochondrial hydroxylase necessary for biosynthesis of ubiquinone, an endogenous antioxidant and cofactor in cellular redox pathways. Ubiquinone is present in all membranes and is responsible for the hydroxylation of 5-demethoxyubiquinone to 5-hydroxyubiquinone in the mitochondrial electron transport chain.48 The cardiovascular system and mCLK1 Mutations causing partial or complete inactivation of mCLK1 are associated with delayed aging. Mice lacking mCLK1 are also protected from ischemia-reperfusion injuries, suggesting a role in vascular function and ischemic response. Studies show that partial loss of function impedes but does not stop certain developmental processes, including embryogenesis and the cell cycle (http://emboj.embopress.org/content/18/7/1783).48 Oxidative stress and mCLK In mCLK haplotype mice, aging is delayed, despite increased generation of mitochondrial ROS. As well as protection from this oxidative stress, haplotype mice also have enhanced immunity and suffer limited damage from other challenges. Biomarkers of oxidative stress in aging are also attenuated in the haplotype mouse.

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LKB1

SIRT1 +Mitochondrial function +Stress resistance +Autophagy

UCP2 IRS2

TSC1/2 Nampt NAD+ AMPK +Mitochondrial function +Lipid metabolism +Autophagy

FOXO IGF1 -Metabolism +Stress resistance

ERK1/2

AKT

TSC1/2

TOR/mTOR +Metabolism +Protein synthesis -Stress resistance -Autophagy

FIG. 7 Interactions of the longevity network. The longevity network principally refers to positive and negative feedback mechanisms between four genes: SIRT1, IGF1, AMPK, and mTOR. The signaling between these genes regulates biologic aging and numerous age-related diseases, including cardiovascular diseases. At the end of each line, an arrowhead indicates positive regulation/stimulation, and a round end indicates negative regulation or inhibition. SIRT1, sirtuin 1; TSC1/2, tuberous sclerosis 1/2; UCP2, mitochondrial uncoupling protein 2; IRS2, insulin receptor substrate 2; FOXO, O class forkhead box transcription factor; IGF1, insulin-like growth factor 1; AKT, protein kinase B; TOR/mTOR, target of rapamycin/mammalian target of rapamycin; ERK1/2, extracellular signal-regulated kinases 1/2; AMPK, adenosine monophosphate-activated protein kinase; Nampt, nicotinamide phosphoribosyltransferase; NADH, nicotinamide adenine dinucleotide; LKB, liver kinase B. Modified from North BJ, Sinclair DA. The intersection between aging and cardiovascular disease. Circ Res 2012;110:1097–108. https://doi.org/10.1161/CIRCRESAHA.111.246876.

Seemingly, there is an initial increase in oxidative stress, with a subsequent higher protective capacity. A similar phenotype, without the longer life span, is observed in mice lacking antioxidant enzymes such as superoxide dismutase.48, 49 This marks mCLK-modulated mitochondrial oxidative stress as a “safe” process to alter within the biologic aging framework. Given the enhanced immunity and oxidative stress resistance, mCLK is currently a valuable target for investigation.48, 49 In light of the aforementioned, the mCLK knockout mouse provides a genetic model to study mitochondrial oxidative stress in age-related cardiovascular decline. Work thus far is indicative of an increasingly important role for mitochondrial metabolism in cardiovascular aging.

Catalase Catalase contains four porphyrin heme groups to facilitate degradation of hydrogen peroxide. Because of a high specific reactivity and easily detectable outcome, catalase is used analytically and industrially (e.g., in classifying bacteria).50 Physiologically, catalase is a very common redox enzyme in nearly all aerobic organisms that protects the organism from oxidative stress. Despite this

important function, catalase deficiency produces a largely normal phenotype. Animals and humans with acatalasia are only slightly more sensitive to oxidative stress.51 The cardiovascular system and catalase When catalase is overexpressed in mitochondria, agerelated cardiac damage is reduced, and life span is increased by 20%.52 Transgenic mice are resistant to hypertrophy, fibrosis, angiotensin II-mediated mitochondrial damage, and Gq-α subunit-mediated heart failure.52, 53 In an ex vivo study of rat hearts, cardiac dysfunction occurs with ischemia and ischemia-reperfusion. When treated with catalase (and cofunctioning superoxide dismutase), the ischemia-reperfusion damage is ameliorated. Other abnormalities from ischemia alone were also ameliorated (e.g., disruption of sodium/potassium ATPase, sodiumcalcium exchange, and calcium uptake and release).54 Oxidative stress and catalase Typically, catalase is a large contributor to neutralizing metabolically by-produced hydrogen peroxide to water and oxygen, thus preventing accumulation of hydroxyl

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radicals. It is theorized that other redox proteins, such as superoxide dismutase, will compensate when catalase levels are low.54 However, catalase also interacts with many important pathways related to oxidative balance (e.g., host and pathogen defense and alcohol metabolism). Catalase also forms part of myocardial local redox defense systems against systemic oxidative stress (e.g., in obesity or insulin resistance). Under such conditions, blocked angiotensin receptors increase antioxidant enzyme activity by 50%–70%, with catalase having a marked increase.55 A significant role for catalase has also been demonstrated in oxidative stress-induced cardiac remodeling. Mice with cardiac-specific Gq-α subunit overexpression provide a model for structural changes in the cardiovascular system. In their system, oxidative stress also increases at a faster than normal rate. The increase in oxidative stress causes dilated cardiomyopathy, which progresses to heart failure. When Gq-α subunit overexpressing mice are crossbred with cardiac-specific catalase overexpressing mice, there is a reduction in age-related and oxidative stress-related structural changes. Myocyte hypertrophy, apoptosis, and heart failure were all prevented in crossbred mice overexpressing both the Gq-α subunit and catalase in the heart. This occurred without affecting the initial oxidative stress phenotype of Gq-α subunit overexpressing mice.56

29

association of klotho single-nucleotide polymorphisms (SNPs) with age-related cardiovascular decline.5, 60 Oxidative stress and Klotho In vitro and in vivo research has provided evidence of klotho’s potential to increase the endothelial layer’s resistance to oxidative stress. This is achieved by maintaining signals for nitric oxide production. Defective klotho signaling also affects the heart, leading to fatal sinoatrial node dysfunction under stress.6, 61

Pituitary transcription factor 1 and prophet of pituitary transcription factor 1 Pituitary transcription factor 1 (PIT1) is a pituitaryspecific transcription factor responsible for normal pituitary development and for expression of hormones regulating mammalian growth.62 Homeobox protein prophet of PIT1 (PROP1), another pituitary transcription factor, possesses transcriptional activation properties and DNA-binding properties. PROP1 expression leads to the development of specialized pituitary cells. The knockout of PIT1 in Snell/ Ames dwarf mice inhibits the development of such anterior pituitary cells, in turn disturbing expression of other signaling peptides and contributing to the risk of diseases.62

Klotho is a transmembrane protein, related to β-glucuronidases, that is highly expressed in specific kidney and brain regions. Klotho hydrolyses steroid β-glucuronides and partially regulates systemic glucose metabolism and insulin sensitivity.57 Overexpression of klotho delays aging, whereas deficiency results in a phenotype resembling human accelerated aging.58, 59

Cardiovascular disease and PIT1/PROP1 Mostly what is known about PIT1 and PROP1 is their effects on biologic aging and growth. Patients deficient in PIT1 and PROP1 typically have growth hormone deficiency. PROP1 deficiency also causes hypogonadism and deficiency of prolactin and thyroid-stimulating hormone.62 Interestingly, Snell/Ames dwarf mice with PROP1 deficiency and delayed aging also exhibit lower cardiac collagen content and smaller cardiomyocytes. These properties may be beneficial with regard to preserving cardiovascular health in aging.63

The cardiovascular system and Klotho Klotho expression maintains cardiovascular health. In fact, low klotho expression is thought to be a leading cause of vascular degeneration in chronic renal failure patients. Deficiency is also associated with arteriosclerosis, impaired angiogenesis, and impaired endotheliumdependent vasodilation.5 Soluble klotho is formed when the extracellular domain is shed into the circulation; its levels decline with age. Cell surface binding sites with which soluble klotho interacts remain unknown. It is, however, known that klotho binding results in perturbed intracellular insulin/IGF1 signaling. This is proposed as a mechanism for klotho’s implication in healthy aging, as the aging phenotype of klotho-deficient mice is averted by perturbing the insulin/IGF1 signaling cascade. This explains the

Oxidative stress and PIT1/PROP1 In Snell/Ames dwarf mice, where the PIT1/PROP1 signaling is perturbed, there is an altered response to oxidative stress. This altered response is thought to increase resistance to oxidative damage and contribute to healthy aging in this mouse.64 The dwarf mouse is known to be particularly resistant to mitochondrial oxidative stress. This was investigated by inducing mitochondrial oxidative stress through inhibition of mitochondrial complex II with 3-nitropropionic acid (3-NPA) treatment. The activator protein 1 (AP-1) transcription factor, which regulates transcriptional responses to numerous stimuli, contains c-Jun family proteins. Phosphorylation of Ser63 and Ser73 residues on the c-Jun protein is necessary to mediate apoptotic and/or proliferative responses to oxidative stress. In the dwarf

Klotho

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mouse, after generation of mitochondrial oxidative stress by 3-NPA treatment, there is a lack of c-Jun Ser63 phosphorylation. This contrasts with the wild type rapid and robust phosphorylation of Ser63 and Ser73 residues. This mechanism allows cell survival in the face of mitochondrial oxidative stress in the dwarf mouse.64

p66Shc The Shc locus regulates several metabolic processes in mammals. Three splice variants are coded with molecular masses of 46, 52, and 66 kDa; they each carry a Src homology 2 domain, a collagen-homology region and a phosphotyrosine-binding domain. The p66 splice variant has a unique N-terminal region with redox enzyme properties. It is actively involved in generating mitochondrial ROS. This N-terminal region is also part of the signaling cascade that translates oxidative signals to apoptosis.5, 65 The cardiovascular system and p66Shc Knockout of p66Shc delays aging and decreases generation of ROS, cardiac progenitor cell senescence, necrosis, and DNA damage; vascular endothelial cell resistance to oxidative stress is increased.66 Loss of p66Shc maintains left ventricular volume, reduces heart failure, and protects high-fat fed mice from atherogenesis.66 In humans, p66Shc expression increases with age, under basal and disease-related conditions.67 Generally speaking, p66Shc is a mediator of many cellular processes, namely, apoptosis and stress response to maintain cardiovascular function. Oxidative stress and p66Shc A mechanistic model of p66Shc is that it oxidizes cytochrome c, preventing it from reducing oxygen radicals to water. Cytochrome c is at the final steps of mitochondrial oxidative phosphorylation. The presence of p66Shc at this point diverts electron flow to produce hydrogen peroxide rather than water. Hydrogen peroxide then opens the mitochondrial permeability transition pore (PTP) that increases mitochondrial membrane permeability to ions, solutes, and water. This increased influx swells and ruptures mitochondria, releasing molecules such as cytochrome c into the cytosol. Cytochrome c acts as a proapoptotic factor in the cytosol. Therefore, p66Shc plays a vital role in mediating cellular generation of oxidants (e.g., hydrogen peroxide) and cellular apoptotic responses through downstream signaling.9 See Fig. 8 for the process through which p66Shc induces mitochondrial rupture and cellular apoptosis. What remains unclear is the signaling between extracellular, intracellular, exogenous, and endogenous oxidative stress and p66Shc. It is hypothesized that p66Shc traffics between cytosolic and mitochondrial compartments following phosphorylation by protein kinase C-β (PKCB)

in response to oxidative stress. Entry of p66Shc disrupts calcium signaling, thus rearranging and fragmenting the mitochondrial matrix, allowing the pathway of oxidative stress and apoptosis to continue. This provides a convenient explanation for p66Shc knockout animals having similar resistance to apoptotic signals responding to exogenous and endogenous oxidative stress. This creates a third role for p66Shc as an intermediary signaling molecule and generating oxidative stress and regulating apoptosis.9

Recent advances in cardiovascular disease, aging and oxidative stress Deciphering the mechanisms of and complex responses to oxidative stress is indeed a challenging field as ROS and RNS are highly volatile and reactive and are found at low concentrations in the organism. Due to the diversity and the complexity of the responses observed in the context of oxidative stress, the development datadriven screening approaches such as high-throughput sequencing, multiomics, and systems biology will lead to a better understanding of the dynamic and complex networks.68–71 For example, recent -omics studies have shown a critical role for epigenomic remodeling in response to oxidative stress and/or aging.72 Methylome analysis revealed aging-associated changes in whole blood cells of healthy subjects.73 It has notably been shown that oxidative stress induces changes in the pool of promoter-associated antisense long noncoding RNAs (lncRNAs) providing new insights of the transcriptional responses to oxidative Chromatin-immunoprecipitation (ChIP)stress.74 sequencing strategy was applied to assess the network of Nrf2-regulated genes.75 Moreover, extensive proteome remodeling occurs in aging animals76, 77 alongside numerous posttranslational modifications (PTMs; e.g., acetylation, succinylation, and oxidation of cysteine residues). Innovative mass spectrometry strategies applied 13 C-glucose, D3-acteate, and cysteine isotopes to study incorporation of acetyl groups into proteins and to identify the acetylome and thiol oxidation proteome. These studies revealed distinct cysteine oxidation patterns of metabolic proteins in response oxidative stress.78, 79 The social and economic burden of diseases related to oxidative stress, notably physiological decline with aging and cardiovascular disease, have led to targeting major actors in oxidative stress to slow down the aging process and its related pathologies. In this context, dietary manipulations such as intermittent fasting and caloric restriction (CR) have been shown to be extremely effective in improving health and life span in several organisms including primates.80 In humans, first clinical trials in obese and nonobese subjects lead to promising results with a decrease of the oxidative stress markers and an improvement of the cardiovascular and general health.81–83

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Oxidative stress

PKCb

Increases membrane permeability leading to mitochondrial overload, rupture and the release of proapoptotic molecules into the cytosol

p66

Cytosol p66 p66 inhibitory complex

PTP Mitochondria p66 H2O2

p66 e–

Ubiquitin

H+

CytC

e–

Oxidative phosphorylation

e– I NADH

NAD+

IV

III

II

O2 FADH2

FAD

H2O

+

Shc

FIG. 8 The role of p66 in apoptosis. Oxidative stress stimulates protein kinase C-β that phosphorylates p66Shc. p66Shc can then traffic into the mitochondria where it is bound to an inhibitory complex. Proapoptotic stimuli destabilize p66Shc, allowing it to oxidize cytochrome c between the third and fourth steps of oxidative phosphorylation. This catalyzes the production of hydrogen peroxide from oxygen. Hydrogen peroxide induces the opening of the mitochondrial permeability transition pore, leading to an increase in mitochondrial membrane permeability to ions, solutes, and water. Mitochondria then swell and rupture, releasing proapoptotic factors into the cytosol. Modified from Cosentino F, Francia P, Camici GG, et al. Final common molecular pathways of aging and cardiovascular disease: role of the p66Shc protein. Arterioscler Thromb Vasc Biol 2008;28:622–8.

Therefore not only molecular and cellular mechanisms induced by CR (protein deacetylation and autophagy) but also tools to mimic these effects (calorie restriction mimetics, CRMs) are of great interest.84 Indeed, different strategies to modulate sirtuin activity are being studied.85 For instance, the selective activation of Sirt1 with chemical compounds enhances numerous antioxidant defense mechanisms and thus increases the life span of mice fed a standard diet.86 Furthermore, resveratrol that is a dietary polyphenol is a cardiometabolicprotective agent in response to high-fat and high-sucrose diet.87 Resveratrol is known to signal through Sirt1 and AMPK pathways. To conclude, multiomics strategies give unprecedented insights in the field of oxidative stress, aging, and cardiovascular health, leading to a not only better understanding of the networks involved in the physiology of these processes but also new potential biomarkers of oxidative stress. Innovative therapies based on CR are under development to combat physiological decline in aging and oxidative stress.

Applications to other diseases and conditions Several studies have demonstrated the importance of oxidative stress in the progression of age-related diseases, alongside cardiovascular pathologies. These conditions are more thoroughly discussed in other chapters of this

edition; however, in this section, we present an overview of the role of oxidative stress in different pathological contexts. Neurodegenerative pathologies: Accumulation of oxidative damages associated with aging occurs in most organs, particularly in the brain and the nervous system.88 The human brain requires approximately 20% of the organism’s oxygen to function rending this organ susceptible to oxidative stress. Notably, Parkinson’s disease, Alzheimer’s disease, and Huntington disease are neurodegenerative pathologies associated with the accumulation and aggregation of specific proteins in the brain. It has been shown that the formation of these aggregates is caused by and causes the production of ROS.89 Metabolic conditions: Oxidative stress plays a key role in the progression of type 2 diabetes (T2D) and its complications, notably cardiovascular pathologies. T2D is characterized by hyperglycemia associated with an inadequate insulin secretion and insulinoresistance. Obesity is a risk factor for the development of T2D. It has been shown that oxidative stress precedes the onset of insulinoresistance induced by high-fat diet.90 Moreover, the production of mitochondrial ROS characterized by dynamic change in mitochondrial morphology91 was identified as the main cause of the glucose-mediated vascular damages in the context of T2D.92 Autoimmune diseases: In the context of autoimmunity, the role of ROS is complex. On the one hand, oxidative stress can be deleterious in several autoimmune

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pathologies such as rheumatoid arthritis (RA), multiple sclerosis, and psoriasis.93 Oxidative stress is a pathogenic hallmark of RA. ROS and RNS can act as mediators of the inadequate activation of the immune system. For instance, there is a production of hypochlorous acid by myeloperoxidase, an enzyme found in neutrophil granules in RA patients and consequently promoting oxidative stress.94 Moreover, PTMs of proteins related to ROS represent neoepitopes and can give rise to autoantibodies, notably against type II collagen in the context of RA.95 On the other hand, oxidative stress can be protective.96, 97 It is indeed suggested that ROS can limit inflammation and autoimmunity by modulating the activation and reactivity of T cells during antigen presentation. Cancer: ROS have been identified as actors of all the processes related to tumor development (transformation, survival, proliferation, invasion, metastasis, and angiogenesis).98 Several environmental factors such as pollutants, smoking, and alcohol are source of oxidative stress. It can induce DNA damages, and aberrant DNA repair can lead to tumorigenesis.99 Finally, oxidative stress is an important field of interest due to its role in several pathologies. Antioxidant therapies are attractive, but many remain inefficient due to the dual role of oxidative stress, which is protective in the context of bacterial infection and deleterious for under conditions of sterile inflammation, notably aging, autoimmunity, or metabolic diseases.

Summary points • Cardiovascular disease is a burden on the population, in terms of lives lost and the economy. • The aging portion of the population is increasing and is more likely to suffer the effects of cardiovascular decline. • Reduced cardiac antioxidant enzyme activity and increased generation of oxidative stress with aging contributes significantly to the risk of cardiovascular disease. • The telomere theory states that telomere attrition, increases with oxidative stress and with age, significantly contributes to cardiovascular decline. • The cardiovascular system is especially sensitive to mitochondrial generation of ROS because cardiomyocytes are rich in mitochondria to meet the continuous and lifelong demand for energy and oxygen. • On the cellular level, mitochondrial oxidative stress is a significant mediator of age-related cardiovascular decline. • SIR2, IGF1, mTOR, and AMPK are key genes in the longevity network that regulate oxidative stress and associated changes to cardiovascular function.

• p66Shc is a powerful mediator of cellular oxidative balance. It functions as a signaling peptide for oxidative stress, as a contributor to mitochondrial generation of reactive oxygen species and as an intermediary signaling molecule in apoptosis. • Novel -omics methods have come to the forefront to decipher the complex causes of and responses to oxidative stress. • High-density and data-driven work will define novel therapeutic targets and lead to the discovery of important biomarkers of oxidative stress and cardiovascular disease. • Calorie restriction, calorie restriction mimetics, and intermittent fasting are promising preventative approaches currently studied in aging and cardiovascular disease.

References 1. Nichols M, Townsend N, Luengo-Fernandez R, et al. European cardiovascular disease statistics 2012. Brussels/Sophia Antipolis: European Heart Network/European Society of Cardiology; 2012. 2. Centre for Disease Control. Chronic disease prevention and health promotion. Available from: http://www.cdc.gov/chronicdisease/ resources/publications/AAG/dhdsp.htm; 2010. Accessed 30 August 2013. 3. Scarborough P, Wickramasinghe K, Bhatnagar P, Rayner M. Trends in coronary heart disease 1961–2011. London: British Heart Foundation; 2011. 4. Kaasik A, Kuum M, Wilding J, et al. Mitochondria as a source of mechanical signals in cardiomyocytes. Cardiovasc Res 2010; 87:83–91. https://doi.org/10.1093/cvr/cvq039. 5. North BJ, Sinclair DA. The intersection between aging and cardiovascular disease. Circ Res 2012;110:1097–108. https://doi.org/ 10.1161/CIRCRESAHA.111.246876. 6. Samani NJ, van der Harst P. Biological ageing and cardiovascular disease. Heart 2008;94:537–9. https://doi.org/10.1136/ hrt.2007.136010. 7. Mehdi MM, Rizvi SI. N,N-dimethyl-p-phenylenediamine dihydrochloride-based method for the measurement of plasma oxidative capacity during human aging. Anal Biochem 2013;436:165–7. https://doi.org/10.1016/j.ab.2013.01.032. 8. Antelmi I, de Paula RS, Shinzato AR, et al. Influence of age, gender, body mass index, and functional capacity on heart rate variability in a cohort of subjects without heart disease. Am J Cardiol 2004;93:381–5. 9. Cosentino F, Francia P, Camici GG, et al. Final common molecular pathways of aging and cardiovascular disease: role of the p66Shc protein. Arterioscler Thromb Vasc Biol 2008;28:622–8. 10. van der Loo B, Labugger R, Skepper JN, et al. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med 2000;192:1731–44. 11. Demissie S, Levy D, Benjamin EJ, et al. Insulin resistance, oxidative stress, hypertension, and leukocyte telomere length in men from the Framingham Heart Study. Aging Cell 2006;5:325–30. 12. Fitzpatrick AL, Kronmal RA, Gardner JP, et al. Leukocyte telomere length and cardiovascular disease in the cardiovascular health study. Am J Epidemiol 2007;165:14–21. 13. Cawthon RM, Smith KR, O’Brien E, et al. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet 2003;361:393–5.

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71. Wang RS, et al. Systems biology approaches to redox metabolism in stress and disease states. Antioxid Redox Signal 2018;29(10):953–72. 72. Guillaumet-Adkins A, et al. Epigenetics and oxidative stress in aging. Oxidative Med Cell Longev 2017;2017:9175806. 73. Kananen L, et al. Aging-associated DNA methylation changes in middle-aged individuals: the Young Finns study. BMC Genomics 2016;17:103. 74. Giannakakis A, et al. Contrasting expression patterns of coding and noncoding parts of the human genome upon oxidative stress. Sci Rep 2015;5:9737. 75. Malhotra D, et al. Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis. Nucleic Acids Res 2010;38(17):5718–34. 76. Walther DM, Mann M. Accurate quantification of more than 4000 mouse tissue proteins reveals minimal proteome changes during aging. Mol Cell Proteomics 2011;10(2). M110 004523. 77. Walther DM, et al. Widespread proteome remodeling and aggregation in aging C. elegans. Cell 2015;161(4):919–32. 78. Kori Y, et al. Proteome-wide acetylation dynamics in human cells. Sci Rep 2017;7(1):10296. 79. van der Reest J, et al. Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress. Nat Commun 2018; 9(1):1581. 80. Mattison JA, et al. Caloric restriction improves health and survival of rhesus monkeys. Nat Commun 2017;8:14063. 81. Ravussin E, et al. A 2-year randomized controlled trial of human caloric restriction: feasibility and effects on predictors of health span and longevity. J Gerontol A Biol Sci Med Sci 2015;70(9):1097–104. 82. Redman LM, et al. Metabolic slowing and reduced oxidative damage with sustained caloric restriction support the rate of living and oxidative damage theories of aging. Cell Metab 2018;27(4):805–15. e4. 83. Most J, et al. Significant improvement in cardiometabolic health in healthy nonobese individuals during caloric restriction-induced weight loss and weight loss maintenance. Am J Physiol Endocrinol Metab 2018;314(4):E396–405. 84. Madeo F, et al. Caloric restriction mimetics against age-associated disease: targets, mechanisms, and therapeutic potential. Cell Metab 2019;29(3):592–610. 85. Grabowska W, Sikora E, Bielak-Zmijewska A. Sirtuins, a promising target in slowing down the ageing process. Biogerontology 2017; 18(4):447–76. 86. Mitchell SJ, et al. The SIRT1 activator SRT1720 extends lifespan and improves health of mice fed a standard diet. Cell Rep 2014; 6(5):836–43. 87. Mattison JA, et al. Resveratrol prevents high fat/sucrose dietinduced central arterial wall inflammation and stiffening in nonhuman primates. Cell Metab 2014;20(1):183–90. 88. Moller P, et al. Aging and oxidatively damaged nuclear DNA in animal organs. Free Radic Biol Med 2010;48(10):1275–85. 89. Kim GH, et al. The role of oxidative stress in neurodegenerative diseases. Exp Neurobiol 2015;24(4):325–40. 90. Matsuzawa-Nagata N, et al. Increased oxidative stress precedes the onset of high-fat diet-induced insulin resistance and obesity. Metabolism 2008;57(8):1071–7. 91. Yu T, Robotham JL, Yoon Y. Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. Proc Natl Acad Sci USA 2006;103 (8):2653–8. 92. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414(6865):813–20. 93. Di Dalmazi G, et al. Reactive oxygen species in organ-specific autoimmunity. Auto Immun Highlights 2016;7(1):11. 94. Strzepa A, Pritchard KA, Dittel BN. Myeloperoxidase: a new player in autoimmunity. Cell Immunol 2017;317:1–8.

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Further reading

95. Strollo R, et al. Autoantibodies to posttranslationally modified type II collagen as potential biomarkers for rheumatoid arthritis. Arthritis Rheum 2013;65(7):1702–12. 96. Hultqvist M, et al. The protective role of ROS in autoimmune disease. Trends Immunol 2009;30(5):201–8. 97. Kienhofer D, Boeltz S, Hoffmann MH. Reactive oxygen homeostasis— the balance for preventing autoimmunity. Lupus 2016;25(8): 943–54. 98. Sosa V, et al. Oxidative stress and cancer: an overview. Ageing Res Rev 2013;12(1):376–90. 99. Bertram C, Hass R. Cellular responses to reactive oxygen speciesinduced DNA damage and aging. Biol Chem 2008;389(3):211–20.

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Further reading Shaik S, Wang Z, Inuzuka H, Wei W. Wang Z, editor. Endothelium aging and vascular diseases, senescence and senescence-related disorders. InTech; 2013 https://doi.org/10.5772/53065. Available from: http://www.intechopen.com/books/senescence-and-senescencerelated-disorders/endothelium-aging-and-vascular-diseases. Sudheesh NP, Ajith TA, Ramnath V, Janardhanan KK. Therapeutic potential of Ganoderma lucidum (Fr.) P. Karst. against the declined antioxidant status in the mitochondria of post-mitotic tissues of aged mice. Clin Nutr 2010;29:406–12. https://doi.org/10.1016/j.clnu. 2009.12.003.

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C H A P T E R

4 Aging, oxidative stress, mitochondrial dysfunction, and the liver Janice G. Lozada-Delgadoa, Carlos A. Torres-Ramosb, Sylvette Ayala-Pen˜aa a

Department of Pharmacology and Toxicology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, United States bDepartment of Physiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, United States

dysfunction seems to play a critical role in this process. Mitochondria not only play a central role in cellular energy production as generators of ATP but also are the main producers of endogenous reactive oxygen species (ROS).4 It has been suggested that the declines in liver function with advancing age may result from the increased generation of mitochondrial ROS. Many liver-associated diseases such as alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and hepatocellular carcinoma may result from the increased generation of ROS by dysfunctional mitochondria.5 The mitochondrial theory of aging postulates that mitochondrial-generated ROS induce the accumulation of somatic mutations in the mitochondrial DNA (mtDNA) that results in impairment of the process of oxidative phosphorylation and ATP production. Not only the prevalence of chronic liver disease is increasing in the elderly population,6 but also the associated morbidity and risk of death are significantly greater in the aged population compared with the young. Therefore understanding the molecular basis of liver disease is important for early diagnosis and treatment. Despite significant progress in the understanding of age-related liver dysfunction and disease, the molecular mechanisms by which aging may damage the liver remain uncertain. Substantial evidence suggests that oxidative stress and mitochondrial dysfunction are important contributors of age-associated liver pathology. In this chapter, we focus on discussing the role of oxidative damage and mitochondrial dysfunction in the pathophysiology of the aging liver.

List of abbreviations 4-HNE 8-oxo-dG ANT AP sites BER GCL Gpx-1 H2O2 MnSOD mtDNA NAFLD NASH NER ROS T2DM TFAM

4-hydroxynonenal 8-oxo-7,8-dihydroguanine adenine nucleotide translocase apurinic/apyrimidinic sites base excision repair glutamate cysteine ligase glutathione peroxidase-1 hydrogen peroxide manganese superoxide dismutase mitochondrial DNA nonalcoholic fatty liver disease nonalcoholic steatohepatitis nucleotide excision repair reactive oxygen species type 2 diabetes mellitus mitochondrial transcription factor A

Introduction Aging is the main risk factor for chronic diseases, as age increases the incidence and progression of pathology including those associated with the liver.1 The liver plays a pivotal role in the process of aging by combining energy metabolism pathways and drug and xenobiotics detoxification.2 The function of the liver declines with age, and liverrelated deaths are also increased, suggesting that aging of the liver may enhance disease susceptibility and predispose the elderly to exacerbated pathology. Aging not only leads to various morphological and structural changes in the liver but also triggers decreases in metabolic function.3 Aging increases the levels of oxidative stress in various organs including the liver, and mitochondrial

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4. Aging, oxidative stress, and the liver

Evidence for age-associated morphological, structural, and functional changes in the liver Aging of the liver manifests as both structural and functional alterations and is evident in humans and rodents. Early ultrasound studies suggest that liver from healthy individuals undergoes a significant 30% reduction in volume with increasing age (reviewed in Ref. 7). Studies in aged mice and rats have also provided evidence of significant declines in hepatic weight similar to those observed in humans.8 Consistent with impaired age-related changes in liver structure, hepatic blood circulation is significantly reduced 30%–50% in rodents and human subjects and correlates with increasing age (reviewed in Ref. 7). In addition, sinusoidal blood flow in livers from aged rats undergoes a significant 30% reduction, which correlates with increased liver sinusoidal endothelial cell dysfunction.9 There is substantial evidence in humans, baboons, and rodents that the liver sinusoidal endothelium and space of Disse undergo ultrastructural changes associated with the process of aging. A significant 40% increase in sinusoid endothelial thickness is observed in rats and baboons, whereas a significant 60% increase is observed in surgical and postmortem human livers from subjects >60 years (reviewed in Ref. 7). Furthermore, electron microscopic analyses show a significant 40%–80% age-related reduction in the number of sinusoidal fenestrations in rat, baboon, and human livers (reviewed in Ref. 7) and in mice.10 Interestingly, early studies show that aging is associated with increases in mitochondrial size in intact hepatocytes11 and in mouse mitochondrial fractions.12 Taken together, these observations support the idea that aging results in morphological and functional alterations in the liver (Fig. 1).

Evidence for age-associated loss of mitochondrial bioenergetics in liver Mitochondrial bioenergetics is essential for maintaining liver function, as these organelles are responsible for producing most of the energy required for appropriate cellular function through the generation of ATP.4 ATP is produced by the oxidation of carbohydrates and fats during oxidative phosphorylation when electrons donated to complexes I (NADH dehydrogenase) and II (succinate dehydrogenase) are sequentially transferred to coenzyme Q and then to complex III, which in turn transfers them to cytochrome c. From cytochrome c the electrons are passed to complex IV (cytochrome oxidase) and finally to molecular oxygen to produce water. While driving the flow of electrons to molecular oxygen, complexes I, II, and IV extrude protons from the matrix to

Aging

Liver

Structural Decreased blood flow Decreased liver mass Increased thickness of the hepatic sinusoids Decreased fenestrations Altered mitochondrial numbers

Functional and metabolic Impaired liver metabolic function Decreased macromolecular synthesis Decreased mitochondrial function Increased oxidative stress Increased oxidative damage

Impaired liver function FIG. 1 Age-associated structural and functional alterations in the liver. During aging a number of structural, functional, and metabolic alterations have been described. These alterations may result in impaired liver function.

the intermembrane space, thus creating a proton current/membrane potential at the inner mitochondrial membrane. The reentry of protons back to the matrix through complex V (ATP synthase) drives the synthesis of ATP from ADP and inorganic phosphate. This flow of electrons through the mitochondrial respiratory chain represents the oxygen consumption rate. Mitochondria that are tightly coupled will generate the maximum levels of ATP when the electron transfer chain is highly efficient at pumping protons out of the inner mitochondrial membrane for use by the ATP synthase for ATP production.4 Substantial evidence in humans and rodents shows that aging induces the loss of mitochondrial function in liver. Livers from normal individuals over 50 years of age show defective mitochondrial respiration mostly associated with a deficiency in complex IV.13 Deficient mitochondrial respiration and decreased ATP production are also evident in liver from aged rats and mice and in isolated human liver mitochondria.14 Isolated liver mitochondria from old rats show a significant 25% and 33% reduction in complexes I and II, respectively, and a concomitant 41% reduction in ATPase activity.8 The activities of complexes I and IV are decreased in liver of senescent rats compared with control animals,15 and loss of mitochondrial membrane potential and increased proton leak are also evident in mitochondria from old rats.16 A significant 30% decrease in mitochondrial membrane potential is observed in intact hepatocytes from old rats as compared with young rats and is associated with increased mitochondrial size.11 Furthermore, mitochondrial membrane potential and the activity of complexes

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Oxidative stress and antioxidant responses in liver aging

I, II, and IV are reduced in liver from aged rats as compared with young animals.15 Reductions in the activity of key mitochondrial metabolic enzymes further emphasize the role of mitochondrial dysfunction in aging of the liver. Studies show that the expression of genes involved in oxidative phosphorylation such as complex I, complex IV, complex V, and cytochrome c was significantly reduced in liver from aged mice.17 Interestingly, liver of mice with extended longevity shows significant increases in the activities of complexes I–IV as compared with wild-type mice.18 Moreover the authors show that liver expression of the adenine nucleotide translocase, the mitochondrial protein complex responsible for fast exchange of ADP/ATP between cytosol and mitochondria, is significantly increased in the long-lived mice. These results suggest that increased mitochondrial function may contribute to longevity in the Ames dwarf mice. The fact that liver from Ames dwarf mice shows increased levels of antioxidant enzymes19 and decreased generation of mitochondrial hydrogen peroxide (H2O2) compared with wild-type mice further supports a major role for mitochondria in aging of the liver. Consistent with this hypothesis a senescenceaccelerated mouse model exhibits significant downregulation of mitochondrial complex I proteins, decreased ATP levels, and liver pathology.20 Collectively the studies discussed earlier show that mitochondrial markers of energy metabolism are altered in liver aging and thus support a role for mitochondrial dysfunction in disease susceptibility and liver pathology during aging (Fig. 1). Despite these significant observations, it remains unclear how aging leads to mitochondrial dysfunction in the liver. A possible mechanism by which aging may contribute to mitochondrial dysfunction and tissue injury is by the induction of oxidative stress.

Oxidative stress and antioxidant responses in liver aging Mitochondria are the principal sources of endogenous ROS, which are generated under physiological conditions as metabolic products of aerobic cellular metabolism. During oxidative phosphorylation, electrons that are donated to oxygen generate the superoxide anion, a short-lived oxygen radical that can be converted to H2O2 by the action of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD/ SOD2), and catalase and glutathione peroxidase convert H2O2 to water. In the presence of iron and other transition metals, H2O2 can be further converted to the highly reactive hydroxyl radical via the Haber-Weiss/Fenton reaction. The generation rates of mitochondrial ROS increase with age, and macromolecules such as nucleic

39

acids, lipids, and proteins are their main targets and may contribute to the characteristic physiological decline observed in aging. Thus mitochondrial dysfunction leads to the overproduction of ROS, which in the aged liver may cause failure of mitochondrial bioenergetics, tissue dysfunction, and pathology. Aging leads to increased generation of ROS in intact hepatocytes from aged rats as compared with young animals and correlates with lowered mitochondrial membrane potential and increased mitochondrial size.11,21 Isolated liver mitochondria from old rats exhibit increased levels of mitochondrial-generated H2O2 with concomitant increases in lipid peroxidation, reduced total antioxidant capacity, and increased mitochondrial dysfunction compared with young animals.22 Aging leads to increased lipid peroxidation and protein carbonylations in liver mitochondria of rats, mice, and nonhuman primates. Significant increases in the levels of protein carbonylations occur in liver from old mice relative to young animals with the antioxidant enzyme Cu/ZnSOD containing the higher levels of protein carbonyls in the old animals.23 Moreover, increased levels of lipid peroxidation products and carbonylated proteins in liver from old nonhuman primates (Macaca mulatta) compared with young monkeys further support the hypothesis that an age-associated increase in oxidative stress may contribute to liver aging.24 Interestingly, dietary restriction, an intervention that extends both average and maximum life span, retards many age-associated diseases and reduces oxidative stress, attenuates protein carbonylations in liver, and correlates with longevity in various mammalian species.25 Old primary hepatocytes fail to induce glutathione peroxidase-1 (Gpx-1) gene expression compared with young cells, suggesting that an altered antioxidant response may contribute to the increased susceptibility of old primary hepatocytes to oxidative stress.26 The liver is the organ with the highest glutathione content and source of secretion to the plasma.27 Hazelton and Lang28 showed significant decreases (20%–30%) in reduced and total glutathione in liver of senescent mice (> 25 months old). The product of the Gpx-1 gene uses glutathione as cofactor in the enzymatic conversion of H2O2 to water. The livers of Gpx-1 mutant mice show increased levels of lipid peroxidation and increased mitochondria H2O2 production. Decreases in the glutathione antioxidant defense system have been described in nonhuman primates where glutathione peroxidase (GSH-Px) activity decreases 65% in old animals (>22 years old) as compared with young animals (< 8 years old) (Fig. 2, Panel A). Moreover, GSH-Px activity negatively correlates with age.24 A significant 65% decrease in total SOD activity is observed in old monkeys versus middle-age animals (Fig. 2, Panel B). Interestingly, old monkeys show a

1. Oxidative stress and aging

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4. Aging, oxidative stress, and the liver

FIG. 2

Age-associated activity of hepatic antioxidant enzymes. (A) Glutathione peroxidase (GHSPx) activity. One unit of GHSPx is equal to 1 mM of NADPH oxidize/min/mg of protein. n ¼ 5 young, n ¼ 4 middle age, and n ¼ 3 old monkeys; *P < .05 versus young. (B) Total SOD activity. Activity was expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50%, which is equal to 1 unit. n ¼ 5 young, n ¼ 2 middle age, and n ¼ 4 old monkeys. *P < .05 versus middle age. (C) Catalase activity. One unit of activity is equal to the moles of H2O2 degraded/min/mg protein. n ¼ 5 young, n ¼ 3 middle age, and n ¼ 3 old monkeys; *P < .05 versus young. From Castro MR, Suarez E, Kraiselburd E, et al. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys. Exp Gerontol 2012;47:29–37, with permission.

significant 39% increase in catalase activity compared with young animals (Fig. 2, Panel C), suggesting that aging modulates the activity of hepatic antioxidant systems. A positive correlation has been observed between 8-oxo-dG levels in mtDNA and high GSSG/GSH ratio in livers from both mice and rats.29 Overall, studies in various aging models suggest that antioxidant defenses may play a central role in liver function during aging. The emerging picture is one in which the liver in young organisms is in a state of balance between the production of oxidants and the antioxidant defenses (Fig. 3). During aging an imbalance occurs due to (1) increased production of oxidants while the level of antioxidant defenses remains relatively stable, and/or (2) a decrease in the levels of antioxidant defenses

occurs while the levels of oxidants remain the same. A combination of both scenarios is also plausible.

Oxidative damage to the nuclear and mitochondrial genomes in liver aging DNA is constantly being damaged by exogenous (xenobiotics) and endogenous (ROS and RNS) agents that induce a variety of DNA lesions such as base modifications, abasic sites, DNA adducts, and single- and double-strand DNA breaks. If these lesions are not repaired, they may lead to mutations, which can be deleterious for the organism. In addition, unrepaired DNA lesions may disrupt processes such as DNA replication

1. Oxidative stress and aging

Oxidative damage to the nuclear and mitochondrial genomes in liver aging

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FIG. 3 Oxidative stress and liver aging. In young animals the liver exerts a balance between oxidant production and antioxidant defenses. During aging, at least two scenarios may occur: (1) the oxidant levels may increase, and/or (2) the antioxidant defenses may decrease. In both instances the net result is an imbalance known as oxidative stress.

and transcription, altering critical physiological processes. A variety of DNA repair mechanisms have evolved to deal with DNA lesions. DNA bulky lesions that alter the DNA structure such as those induced by UV light and some carcinogens are mainly repaired by nucleotide excision repair (NER). Double-strand breaks are repaired by homologous recombination or by nonhomologous end joining. Damage to DNA bases such as alkylation and oxidative modifications are repaired mostly by base excision repair (BER).30 Increases in DNA lesions have been reported in the liver during aging. For example, 8-oxo-dG levels, which are common oxidative DNA lesion mainly repaired by BER, have been shown to increase in liver from aged rats and mice.31,32 Interestingly a significant 4.7-fold increase in mtDNA lesions is detected in liver of old rhesus monkeys (>19 years of age) compared with young (0.6–2 years of age) rhesus (Fig. 4, Panel A), and the increased frequency of lesions significantly correlates with age.24 Moreover, apurinic/apyrimidinic (AP) site levels, another DNA lesion repaired by BER, increase about 1.7-fold in the liver from old rats (24 months old) as compared with young (4 months old) rats.24 The reason for such increase is not fully understood, but two different (but not mutually exclusive) models could be invoked: increased oxidative stress and decreased DNA repair. Decreases in overall BER enzymatic activity (more than 50% decrease) have been observed in the liver from aged mice.33 However, studies designed to determine if aging affects specific steps within BER show a complicated scenario. For example, an age-dependent decrease in DNA polymerase β-levels (one of the DNA polymerases involved in

the repair synthesis step in BER) has been reported.33 However, no age-related changes in nuclear 8-oxo-dG and UDG glycosylase activities have been detected.34 On the other hand an increase in AP endonuclease activity (an enzyme that acts on AP sites as part of BER) in nuclear extracts from liver has been reported, suggesting a response to increased levels of oxidative stress during aging.35 Thus the repair response to DNA damage during aging is not only tissue specific but also damage and subcellular compartment specific (i.e., nuclear vs mitochondrial). The NER protein ERCC1 participates in the incision step five to the DNA lesion. A mouse model with reduced ERCC1 expression (Ercc1/Δ) was used to study liver aging.10 This mouse liver shows pseudocapillarization and premature loss of liver function, a significant reduction in the number of sinusoidal fenestrations and increased levels of lipid peroxidation similar to those found in old wild-type mice. Although most of the DNA lesions accumulating during liver aging may be oxidative in nature and thus associated with BER, NER may be an important backup repair pathway that complements BER, similarly to the studies performed in simple model organisms.30 Mitochondria are unique organelles in the sense that they contain their own genome. MtDNA in higher eukaryotes consists of a 16,569-bp circular molecule, encoding 13 polypeptides of the electron transport chain, 22 tRNAs and 2 rRNAs.36 Strong evidence shows that mtDNA harbors more damage than nuclear DNA during aging, particularly in tissues with high metabolic rates such as the liver and brain.31 Moreover, mtDNA of cells

1. Oxidative stress and aging

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4. Aging, oxidative stress, and the liver

FIG. 4 Mitochondrial DNA damage increases, and its abundance decreases with age in the liver from rhesus monkeys. Total DNA was isolated from liver obtained from 0.6- to 2.0-year-old (infant), 3- to 8-year-old (young), 9- to 17-year-old (middle age), and 19- to 24-year-old (old) monkeys and analyzed by QPCR. (A) Frequency of mtDNA lesions per 10 kb per strand. *P